Compositions for, and methods of, anti-platelet therapy

ABSTRACT

Cocoa extracts which include procyanidin monomers and their oligomers are useful in the modulation of inflammatory pathways, in the maintenance of the vascular health of a mammal and as an antibacterial treatment. The liquid or dry cocoa extracts can be included in foods, food supplements and pharmaceuticals for the inhibition of COX activity, the inhibition of LOX activity, the enhancement of nitric oxide production, the modulation of eicosanoids and endothelin, and the modulation of platelet activity.

REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation application of U.S.application Ser. No. 09/459,171, filed Dec. 10, 1999, allowed, which isa continuation-in-part application of U.S. application Ser. No.08/831,245 filed Apr. 2, 1997, now U.S. Pat. No. 6,297,273, which is acontinuation-in-part application of abandoned U.S. application Ser. No.08/631,661 filed Apr. 2, 1996. The application Ser. Nos. 09/459,171 and08/631,661 are hereby incorporated herein by reference.

FIELD OF THE INVENTION

[0002] This invention relates to medical and nutritional uses ofextracts and products containing cocoa polyphenols including cocoaprocyanidins.

BACKGROUND OF THE INVENTION

[0003] Polyphenols are an incredibly diverse group of compounds(Ferriera et al., ‘Diversity of Structure and Function in OligomericFlavanoids, Tetrahedron, 48:10, 1743-1803, 1992). They widely occur in avariety of plants, some of which enter into the food chain. In somecases they represent an important class of compounds for the human diet.Although some of the polyphenols are considered to be non-nutritive,interest in these compounds has arisen because of their possiblebeneficial effects on health.

[0004] For instance, quercetin (a flavonoid) has been shown to possessanticarcinogenic activity in experimental animal studies (Deshner etal., ‘Quercertin and Rutin as Inhibitors of Azoxymethanol-inducedColonic Neoplasia’, Carcinogenesis, 7:1193-1196, 1991: and Kato et al.,‘Inhibition of 12-O-tetradecanoylphorbol-13-acetate Induced TumorPromotion and Omithine Decarboxylase Activity by Quercitin: PossibleInvolvement of lipoxygenase Inhibition’, Carcinogenesis, 4, 1301-13051983). (+)-catechin and (−)-epicatechin (flavan-3-ols) have been shownin inhibit Leukemia virus reverse transcriptase activity (Chu et al.,Inhibitory Effects of Flavonoids on Maloney Murine Leukemia VirusReverse Transcriptase Activity, J. of Natural Products, 55:2, 179-183,1992). Nobotanin (an oligomeric hydrolyzable tannin) has also been shownto possess anti-tumor activity (Okuda et al., ‘Molecular Structures andPharmacological Activities of Polyphenols—Oligomeric HydrolyzableTannins and Others'—Presented at the XVIth International Conference ofthe Groupe Polyphenols, Lisbon, Portugal, July 13-16, 1992). Statisticalreports have also shown that stomach cancer mortality is significantlylower in the tea producing districts of Japan. Epigallocatechin gallatehas been reported to be the pharmacologically active material in greentea that inhibits mouse skin tumors (Okuda et al, ‘Molecular Structuresand Pharmacological Activities of Polyphenols—Oligomeric HydrolyzableTannins and others. Presented at the XVIth International Conference ofthe Groupe polyphenols, Lisbon, Portugal, 1992). Osakabe et al. (JP7274894 “Food and Drink For Preventing Gastric Ulcers—ContainsAntioxidation Substance Extracted from Cacao Beans using Hot Water orEthanol” October 1995; JP 7213251 “Method on Manufacturing anAntioxidation Substance and a Health Food or Drink Item Containing anAntioxidation Substance” August 1995) have reported that the antioxidantproperties of cocoa bean extract, thought to contain epicatechin andanalogous compounds, are responsible for inhibiting formation of gastriculcers in rats. Ellagic acid has also been shown to possessanticarcinogen activity in various animal tumor models (Boukharta etal., Efficacy of Ellagitannins and Ellagic Acid as CancerChemopreventive Agents—Presented at the XVIth International Conferenceof the Groupe Polyphenols, Lisbon, Portugal, Jul. 13-16, 1992). Lastly,proanthocyanidin oligomers have been reported by the KikkomanCorporation for use as antimutagens (‘Antimutagenic Agent ContainingProanthocyanidin Oligomer Preferably Having Flavan-3-ol-diol Structure’JP 04190774A, Jul. 7, 1992). Indeed, the area of phenolic compounds infoods and their modulation of tumor development in experimental animalmodels has been recently presented to the 202nd National Meeting of TheAmerican Chemical Society (Phenolic Compounds in Foods and Their Effectson Health II. Antioxidants & Cancer Prevention, Huang, M.-T., Ho, C.-T.,and Lee, C. Y. editors, ACS Symposium Series 507, Am. Chem. Soc.,Washington, D.C. 1992).

SUMMARY OF THE INVENTION

[0005] It has been surprisingly discovered that cocoa extract, andcompounds therefrom, have anti-tumor, anti-cancer or antineoplasticactivity or, is an antioxidant composition or, inhibits DNAtopoisomerase II enzyme activity or, is an antimicrobial or, is acyclo-oxygenase and/or lipoxygenase modulator or, is a NO or NO-synthasemodulator or, is a blood or in vivo glucose modulator.

[0006] Accordingly, the present invention provides a substantially purecocoa extract and compounds therefrom. The extract or compoundspreferably comprises polyphenol(s) such as polyphenol(s) enriched withcocoa procyanidin(s), such as polyphenols of at least one cocoaprocyanidin selected from (−) epicatechin, (+) catechin, procyanidinB-2, procyanidin oligomers 2 through 12, preferably 2 through 4 or 4through 12, more preferably 3 through 12, and most preferably 5 through12, procyanidin B-5, procyanidin A-2 and procyanidin C-1.

[0007] The present invention also provides an anti-tumor, anti-cancer orantineoplastic or antioxidant or DNA topoisomerase II inhibitor, orantimicrobial, or cyclo-oxygenase and/or lipoxygenase modulator, or anNO or NO-synthase modulator, or blood or in vivo glucose modulatorcomposition comprising a substantially pure cocoa extract or compoundtherefrom or synthetic cocoa polyphenol(s) such as polyphenol(s)enriched with procyanidin(s) and a suitable carrier, e.g., apharmaceutically, veterinary or food science acceptable carrier. Theextract or compound therefrom preferably comprises cocoa procyanidin(s).The cocoa extract or compounds therefrom is preferably obtained by aprocess comprising reducing cocoa beans to powder, defatting the powderand, extracting and purifying active compound(s) from the powder.

[0008] The present invention further comprehends a method for treating apatient in need of treatment with an anti-tumor, anti-cancer, orantineoplastic agent or an antioxidant, or a DNA topoisomerase IIinhibitor, or antimicrobial, or cyclo-oxygenase and/or lipoxygenasemodulator, or an NO or NO-synthase modulator, or blood or in vivoglucose modulator comprising administering to the patient a compositioncomprising an effective quantity of a substantially pure cocoa extractor compound therefrom or synthetic cocoa polyphenol(s) or procyanidin(s)and a carrier, e.g., a pharmaceutically, veterinary or food scienceacceptable carrier. The cocoa extract or compound therefrom can be cocoaprocyanidin(s); and, is preferably obtained by reducing cocoa beans topowder, defatting the powder and, extracting and purifying activecompound(s) from the powder.

[0009] Anti-cancer, anti-tumor or antineoplastic or, antioxidant, DNAtopoisomerase II enzyme inhibiting, antimicrobial, cyclo-oxygenaseand/or lipoxygenase modulator NO— or NO-synthase and blood or in vivoglucose modulating activities, or compositions containing the inventivecocoa polyphenols or procyanidins can be prepared in accordance withstandard techniques well known to those skilled in the pharmaceutical orfood science or veterinary art(s).

[0010] Such compositions can be administered to a subject or patient inneed of such administration in dosages and by techniques well known tothose skilled in the medical, nutritional or veterinary arts taking intoconsideration such factors as the age, sex, weight, and condition of theparticular subject or patient, and the route of administration. Thecompositions can be co-administered or sequentially administered withother antineoplastic, anti-tumor or anti-cancer agents, antioxidants,DNA topoisomerase II enzyme inhibiting agents, or cyclo-oxygenase and/orlipoxygenase, blood or in vivo glucose or NO or NO-synthase modulatingagents and/or with agents which reduce or alleviate ill effects ofantineoplastic, anti-tumor, anti-cancer agents, antioxidants, DNAtopoisomerase II enzyme inhibiting agents, cyclo-oxygenase and/orlipoxygenase, blood or in vivo glucose or NO or NO-synthase modulatingagents; again, taking into consideration such factors as the age, sex,weight, and condition of the particular subject or patient, and, theroute of administration.

[0011] Further, the invention also comprehends a kit wherein the activecocoa extract is provided. The kit can include a separate containercontaining a suitable carrier, diluent or excipient. The kit can alsoinclude an additional anti-cancer, anti-tumor or antineoplastic agent,antioxidant, DNA topoisomerase II enzyme inhibitor or antimicrobial, orcyclo-oxygenase and/or lipoxygenase, NO or NO-synthase or blood or invivo glucose modulating agent and/or an agent which reduces oralleviates ill effects of antineoplastic, anti-tumor or anti-canceragents, antioxidant, DNA topoisomerase II enzyme inhibitor orantimicrobial, or cyclo-oxygenase and/or lipoxygenase, NO or NO-synthaseor blood or in vivo glucose modulating agents for co- orsequential-administration. The additional agent(s) can be provided inseparate container(s) or in admixture with the active cocoa extract.Additionally, the kit can include instructions for mixing or combiningingredients and/or administration.

[0012] A cocoa polyphenol-containing composition, which is useful formodulating inflammatory pathways in a mammal, for maintaining vascularhealth, and as an antibacterial treatment, comprising a cocoa ingredientcontaining the cocoa polyphenols and optionally a carrier, diluent, orexcepient. The composition is useful as a food, a dietary supplement, ora pharmaceutical. The food or dietary supplement can be a beverage or anelixir (i.e., ethanol extract), or a powder.

[0013] The cocoa polyphenols can be from ingredients or preparedsynthetically. They can be present in cocoa ingredients. They can beextracted from cocoa beans, cocoa nibs, or cocoa ingredients such aschocolate liquor, partially defatted cocoa solids, and/or fully defattedcocoa solids. The cocoa procyanidins are monomers and/or oligomers ofepicatechin and catechin. The oligomers include dimers throughoctadecamers.

[0014] When extracted from cocoa beans, cocoa nibs, cocoa nib fractions,chocolate liquor, partially defatted cocoa solids, and/or fully defattedcocoa solids, a solvent which dissolves the cocoa polyphenols is used.Suitable solvents include water, methanol, ethanol, acetone, ethylacetate, or mixtures thereof. Preferred solvents are mixtures of waterand methanol or acetone. When water is used as the solvent, it ispreferable if it is slightly acidified. In some cases the extract ispurified, for example by removal of the caffeine and/or theobromine, andthen further purified by gel permeation chromatography and/or highpressure liquid chromatography. During the high pressure liquidchromatography, the extract can be fractionated into monomeric andoligomeric fractions containing at least 50% by weight of the monomersor specific oligomers. When the fractions contain the monomers and loweroligomers (up to and including the tetramer), the fractions containabout 90 to 95% by weight of the particular oligomeric fraction.

[0015] Use of the above composition provides a method for modulating amammalian inflammatory pathway by inhibiting COX activity, formodulating the production of eicosanoids and endothelin, for preventingdiseases (such as bowel disease edema, arthritis, gingivitis orperidontitis) caused by chronic inflammation, for preventing vasculardisease, for enhancing nitric oxide synthesis, for inhibiting LOXactivity, for reducing vasoconstriction, for reducing plateletaggregation, for inhibiting monocyte adhesion, for inhibiting vascularsmooth muscle proliferation associated with vascular disease, forreducing thrombosis, for reducing blood pressure, and for modulatingoxidative stress to prevent associated inflammatory disorders andvascular diseases. Modulation of oxidative stress, for example, bypreventing (LDL) oxidation, is another method of which the abovecomposition can modulate mammalian inflammatory pathways and vascularfunction and disease.

[0016] The products comprising the cocoa polyphenol-containingcomposition are preferably in forms suitable for oral delivery, such astablets, capsules, pills, concentrates, powders, liquids, or foodingredients, food additives or dietary supplements. The tablet maycomprise an effective amount of the cocoa polyphenol-containingcomposition and optionally a carrier, such as sorbitol, lactose,cellulose, or dicalcium phosphate. The capsule may comprise a gelatincapsule containing a predetermined dosage of the cocoapolyphenol-containing composition. The oral delivery product may alsocomprise a dietary supplement nutrient such as dicalcium phosphate,magnesium stearate, calcium nitrate, vitamins, and minerals.

[0017] The compositions comprising the cocoa extract, sub-fractionsthereof or mixtures thereof further comprise a liquid or a solid carriersuitable for use in foods, food supplements or pharmaceuticals. Suchproducts include food and beverage products, in addition to capsule,tablet and pressed powder compositions. For the purposes of thisapplication, the following definitions will enable a clearerunderstanding of what is disclosed and claimed:

[0018] As used herein a “food” is a material consisting essentially ofprotein, carbohydrate and/or fat, which is used in the body of anorganism to sustain growth, repair and vital processes and to furnishenergy. Foods may also contain supplementary substances such asminerals, vitamins and condiments. See Merriam-Webster's CollegiateDictionary, 10^(th) Edition, 1993.

[0019] As used herein, a “pharmaceutical” is a medicinal drug. SeeMerriam-Webster's Collegiate Dictionary, 10^(th) Edition, 1993.

[0020] As used herein, a “Food Supplement” is a product (other thantobacco) that is intended to supplement the diet that bears or containsthe one or more of the following dietary ingredients: a vitamin, amineral, an herb or other botanical, an amino acid, a dietary substancefor use by man to supplement the diet by increasing the total dailyintake, or a concentrate, metabolite, constituent, extract orcombination of these ingredients. See Merriam-Webster's CollegiateDictionary, 10^(th) Edition, 1993. As used on food labels, ‘supplement’means that nutrients have been added in amounts greater than 50% abovethe U.S. RDA (“Understanding Normal and Clinical Nutrition, ThirdEdition”, Eds. Whitney, Cataldo and Rolfes, p. 525).

[0021] The compositions comprising the cocoa extracts, sub-fractionsthereof, or mixtures thereof, are usefull for inhibiting COX activity,inhibiting LOX activity, enhancing nitric oxide production, reducingvasoconstriction, reducing platelet aggregation, inhibiting monocyteadhesion, inhibiting excessive proliferation of vascular smooth muscle,reducing thrombosis, reducing blood pressure and modulating theproduction of eicosanoids and endothelin. Diseases, such as boweldisease, arthritis, edema, gingivitis and peridontitis, which are causedby chronic inflammation, are also prevented.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The following Detailed Description will be better understood byreference to the accompanying drawings wherein:

[0023]FIG. 1 shows a purification scheme for the isolation ofprocyanidins from cocoa.

[0024]FIG. 2a-c is a schematic diagram showing the relationship betweenthe biological activity of the cocoa polyphenols and their utility inthe prevention of coronary heart disease.

[0025]FIG. 3 shows a representative gel permeation chromatogram from thefractionation of crude cocoa procyanidins.

[0026]FIG. 4 shows a representative reverse-phase HPLC chromatogramshowing the separation (elution profile) of cocoa procyanidins extractedfrom unfermented cocoa.

[0027]FIG. 5 shows a representative normal phase HPLC separation ofcocoa procyanidins extracted from unfermented cocoa.

[0028]FIG. 6 shows a MALDI-TOF mass spectrum of cocoa procyanidinoligomers (tetramers to octadecamers).

[0029]FIG. 7 shows the acetylcholine-induced relaxation of NO-relatedphenylephrine-precontracted rat aorta.

[0030]FIGS. 8A and B show the effects of cocoa procyanidin fraction Aand C, respectively, on blood pressure; blood pressure levels decreasedby 21.43% within 1 minute after administration of fraction A, andreturned to normal after 15 minutes, while blood pressure decreased by50.5% within 1 minute after administration of fraction C, and returnedto normal after 5 minutes.

[0031]FIG. 9 shows the effect of cocoa procyanidin fractions on arterialblood pressure in anesthetized guinea pigs.

[0032]FIG. 10 shows the effect of L-NMMA on the alterations of arterialblood pressure in anesthetized guinea pigs induced by cocoa procyanidinfraction C.

[0033]FIG. 11 shows the effect of bradykinin on NO production by HUVEC.

[0034]FIG. 12 shows the effect of cocoa procyanidin fractions onmacrophage NO production by HUVEC.

[0035]FIG. 13 shows the effect of cocoa procyanidin fractions onmacrophage NO production.

[0036]FIG. 14 shows the effect of cocoa procyanidin fractions onmacrophage NO production.

[0037]FIGS. 15A & B show the effects of indomethacin on COX-1 and COX-2activities.

[0038]FIGS. 16A & B show the correlation between the degree ofpolymerization and IC₅₀ vs. COX1/COX2 (μM);

[0039]FIG. 17 shows the correlation between the effects of compounds onCOX—I and COX-2 activities expressed as μM.

[0040]FIG. 18A-V show the IC₅₀ values (EM) of samples containingprocyanidins with COX-1/COX-2.

[0041] FIGS. 19A-D show the effect of phytochemicals on basalendothelial cell synthesis of the prostanoids and endothelins in BAECS.*means significantly different from control at a level of p<0.05.

[0042]FIGS. 20A & B show the effects of cell species on procyanidininduced alterations in EC release of prostacyclin and endothelin.

[0043]FIG. 21A-C shows the effect of cocoa beverage consumption onplatelet surface expression of activated GPIIb-IIIa with and withoutstimulation with weak agonists. Platelet activation marker expression ispresented as Tukey box plots at times zero (white boxes), 2 hours(dotted boxes) and 6 hours (cross-hatched boxes) post consumption ofwater, a caffeine-containing control beverage (caffeine) or a cocoabeverage (cocoa). (A)—percentage of platelets expressing activatedGPIIb-IIIa (PAC1+platelets) without stimulation; (B)—after stimulationwith epinephrine (20 μM); or (C)—with ADP (20 μM). Activated GPIIb-IIIais expressed on the surface of activated platelets. Each box shows the25-75^(th) percentile, the horizontal bar in the box shows the median.The lines outside of the box show the 10^(th) and 90^(th) percentile.Asterisks indicate P<0.05 between zero time and 6 hour time points ofeach respective data set (repeated measure ANOVA on ranks,Student-Newman-Keuls multiple comparison method, n=10 in each).

[0044]FIG. 22A-C shows the effect of cocoa beverage consumption onplatelet surface expression of activated P-selectin with and withoutstimulation with weak agonists. Platelet activation marker expressionpresented as Tukey box plots at times zero (white boxes), 2 hours(dotted boxes) and 6 hours (cross-hatched boxes) post consumption ofwater, a caffeine-containing control beverage (caffeine) or a cocoabeverage (cocoa). (A¹) percentage of platelets expressing P-selectin(CD62P+platelets) without stimulation; (B)—after stimulation withepinephrine (20 μM); or (C)—with ADP (20 μM). P-selectin is expressed onthe surface of activated platelets. Each box shows the 25-75 hpercentile, the horizontal bar in the box shows the median. The linesoutside of the box show the 10^(th) and 90^(th) percentile. Asterisksindicate P<0.05 between zero time and 6 hour time points of eachrespective data set (repeated measure ANOVA on ranks,Student-Newman-Keuls multiple comparison method, n=10 in each).

DETAILED DESCRIPTION

[0045] Monomers comprising procyanidins have the structure:

[0046] Procyanidins include those found in cocoa beans obtained fromTheobroma cacao and various related cocoa species, as well as the genusHerrania and their inter- and intra-genetic crosses.

[0047] Monomers comprising procyanidins include (+)-catechin,(−)-epicatechin and their respective epimers (e.g. (−)-catechin and(+)-epicatechin).

[0048] Synthetic linear and/or branched oligomers having the followingstructures are illustrative of the cocoa procyanidins.

[0049] Linear oligomers where n is an integer from 0 to 16

[0050] Branched oligoniers where A and B are independently oligomersfrom 1 to 15 which total 3-18 in final oligomer.

[0051] In the oligomers n is an integer from 2 through 18, preferably 3through 12, more preferably 5 through 12, and most preferably 5. Theoligomers have interflavan linkages of (4→6) and and/or (4→8). Theoligomers may be represented by the structures above. For the linearoligomer, when x is 0, the oligomer is termed a “dimer”; when x is 1 theoligomer is termed a “trimer”; when x is 2, the oligomer is termed a“tetramer”; when x is 3, the oligomer is termed a “pentamer”; andsimilar recitations may be designated for oligomers having x up to andincluding 16 and higher, such that when x is 16, the oligomer is termedan “octadecamer.” For the branched oligomer, when A or B is 1, theoligomer is termed a “trimer”; with similar recitations such as thosedescribed for the linear oligomers.

[0052] Derivatives of the synthetic cocoa polyphenols include thegallated monomers and oligomers (a method for the preparation of thedimer di-gallate is disclosed in U.S. Ser. No. 09/289,565 filed Apr. 9,1999, the disclosure of which is incorporated by reference), theglycosylated monomers and oligomers, and mixtures thereof. Also includedare metabolites of the monomers and oligomers, including the sulphated,glucoronidated, and methylated forms. Further included are the enzymecleavage products generated by colonic microflora metabolism or internalmammalian metabolism.

[0053] The cocoa extracts are generally prepared by reducing cocoa beansto cocoa powder, defatting the powder, extracting the cocoa polyphenols,and purifying the extract. The cocoa powder can be prepared byfreeze-drying the cocoa beans and pulp, depulping and dehulling thefreeze-dried cocoa beans, and grinding the dehulled beans. The cocoapolyphenols can be extracted from the powder by solvent extractiontechniques. The cocoa extracts can be purified, e.g., to besubstantially pure, for instance, by gel permeation chromatography or bypreparative High Performance Liquid Chromatography (HPLC) techniques orby a combination of such techniques.

[0054] With reference to the extraction and purification of the cocoaextracts, it will be understood that any species of Theobroma, Herraniaor inter- and intra-species crosses thereof may be employed. In thisregard, reference is made to Schultes, Synopsis of Herrania,” Journal ofthe Arnold Arboretum, Vol. XXXIX, pp 217 to 278, plus plates 1 to XVII(1985), Cuatrecases, “Cocoa and Its Allies, A Taxonomic Revision of theGenus Theobroma,” Bulletin of the United States National Museum, Vol.35, page 6, pp. 379 to 613, plus plates 1 to 11 (SmithsonianInstitution, 1964), and Addison, et al., ‘Observations on the Species ofthe Genus Theobroma Which Occurs in the Amazon,” Bol. Tehc. Inst.Agronomico de Nortes, 25(3)(1951). The cocoa procyanidins can beisolated from cocoa or from any species within the Theobroma andHerrania genera. Additionally, Table 4 lists the heretofore neverreported concentrations of the cocoa procyanidins found in Theobroinaand Herrania species and their inter- and intra-species crosses.

[0055] An outline of the purification protocol utilized in the isolationof substantially pure cocoa procyanicins is shown in FIG. 1. The stepsof the purification process are outlined in Examples 1-5. The skilledartisan would appreciate and envision modification in the purificationscheme outlined in FIG. 1 to obtain the active compounds withoutdeparting from the spirit or scope thereof and without undueexperimentation.

[0056] The extracts and/or fractions derived therefrom having activity,without wishing to necessarily be bound by any particular theory, havebeen identified as cocoa polyphenol(s), which include procyanidins.These cocoa procyanidins have to function as NO (Nitric Oxide)modulators, as non-steroidal anti-inflammatory agents, as modulators ofplatelet activation, and as cyclo-oxygenase and/or lipoxygenasemodulators.

[0057] With regard to the cocoa procyanidins, it has been surprisinglyfound that the cocoa procyanidins have discrete activities, and as such,the cocoa procyanidins have broad applicability to the treatment of avariety of disease conditions, discussed herein below.

[0058] COX/LOX-Associated Utilities

[0059] Atherosclerosis, the most prevalent of cardiovascular diseases,is the principle cause of heart attack, stroke and vascular circulationproblems. Atherosclerosis is a complex disease which involves many celltypes, biochemical events and molecular factors. There are severalaspects of this disease, its disease states and disease progressionwhich are distinguished by the interdependent consequences of LowDensity Lipoprotein (LDL) oxidation, cyclo-oxygenase (COX)/lipoxygenase(LOX) activity, eicosanoid and endothelin activities and Nitric Oxide(NO) biochemistry. See FIG. 2.

[0060] Clinical studies have firmly established that elevated plasmaconcentrations of LDL are associated with accelerated atherogenesis. Thecholesterol that accumulates in atherosclerotic lesions originateprimarily in plasma lipoproteins, including LDL. The oxidation of LDL isa critical event in the initiation of atheroma formation and isassociated with the enhanced production of the superoxide anion radical(O₂!-). Oxidation of LDL by O₂!- or other reactive species (e.g., !OH,ONOO!-, lipid peroxy radical, copper ion, and iron based proteins)reduces the affinity of LDL for uptake in cells via receptor mediatedendocytosis. The oxidatively-modified LDLs are then rapidly taken up bymacrophages which subsequently transform into cells closely resemblingthe “foam cells” observed in early atherosclerotic lesions.

[0061] Oxidized lipoproteins can also promote vascular injury throughthe formation of lipid hydroperoxides within the LDL particle. Thisevent initiates radical chain oxidation reactions of unsaturated LDLlipids, thus producing more oxidized LDL for macrophage incorporation.

[0062] The collective accumulation of foam cells engorged with oxidizedLDL from these processes results in early “fatty streak” lesions, whicheventually progress to the more advanced complex lesions ofatherosclerosis leading to coronary disease.

[0063] As discussed generally by Jean Marx at page 320 of Science, Vol.265 (Jul. 15, 1994), each year about 330,000 patients in the UnitedStates undergo coronary and/or peripheral angioplasty, a proceduredesigned to open up blood vessels, e.g., coronary arteries, clogged bydangerous atherosclerotic plaques (atherosclerosis) and thereby restorenormal blood flow. For a majority of these patients, the operation worksas intended. Nearly 33% of these patients (and maybe more by someaccounts), however, develop restenosis, wherein the treated arteriesbecome quickly clogged again. These patients are no better off, andsometimes worse off, than they were before angioplasty. Excessiveproliferation of smooth muscle cells (SMCs) in blood vessel wallscontributes to restenosis. Increased accumulation of oxidized LDL withinlesion SMCs might contribute to an atherogenic-related process likerestenosis, as discussed by Zhou et al., “Association Between PriorCytomegalovirus Infection And The Risk Of Restenosis After CoronaryAtherectomy,” New England Journal of Medicine, 335:624-630, Aug. 29,1996, and documents cited therein. Accordingly, utility of the presentinvention with respect to atherosclerosis can apply to restenosis.

[0064] With regard to the inhibition by the cocoa procyanidins ofcyclo-oxygenases (COX; prostaglandin endoperoxide synthase), it is knownthat cyclo-oxygenases are central enzymes in the production ofprostaglandins and other arachidonic acid metabolites (i.e.,eicosanoids) involved in many physiological processes. COX-1 is aconstitutive enzyme expressed in many tissues, including platelets,whereas COX-2, a second isoform of the enzyme, is inducible by variouscytokines, hormones and tumor promoters. COX-1 produces thromboxane A2,which is involved in platelet aggregation, which in turn is involved inthe progression of atherosclerosis. Its inhibition is the basis for theprophylactic effects on vascular disease.

[0065] The activity of COX-1 and COX-2 is inhibited by aspirin and othernonsteroidal anti-inflammatory drugs (NSAIDs). The gastric side effectsof NSAIDs are believed to be associated with the inhibition of COX-1.Moreover, it has been found that patients taking NSAIDs on a regularbasis have a 40 to 50% lower risk of contracting colorectal cancer whencompared to persons not being administered these type of medications.COX-2 mRNA levels are markedly increased in 86% of human colorectaladenocarcinomas.

[0066] One significant property of COX-2 expressing cell lines is theenhanced expression of genes which participate in the modulation ofapoptosis, i.e., programmed cell death. Several NSAIDs have beenimplicated in increased cell death and the induction of apoptosis inchicken embryo fibroblasts.

[0067] Cellular lipoxygenases are also involved in the oxidativemodification of LDL through the peroxidation of unsaturated lipids. Thegeneration of lipid peroxy radicals contributes to the further radicalchain oxidation of unsaturated LDL lipids, producing more oxidized LDLfor macrophage incorporation.

[0068] Lipoxygenase is a highly specific catalyst for the oxidation ofunsaturated fatty acids containing a cis,cis-1,4-pentadiene system(Tappel et al, in “The Enzymes” Academic Press, New York, N.Y., pp.275-283, 1963). The hydroperoxide products are structurally similar tothose obtained by autoxidation. Lipoxygenase is a nonheme iron protein.The metal, however, is essential for its enzymatic activity (Grossman etal. Methods. Biochem. Anal. 25:303-329, 1979). The mechanism of lipidoxidation is thus distinct from heme containing lipid oxidationcatalysts, i.e., hemoglobin, myoglobin and cytochromes.

[0069] A variety of animal cells (i.e. leukocytes, mast cells and tissuecells) contain specific 5-, 12- and 15-lipoxygenase activitiescatalyzing the formation of leukotrienes and lipoxins. Leukotrienes aregenerated by 5-lipoxygenase from membrane derived arachidonic acid viathe 5-hydroperoxytetraenoic acid intermediate (Samuelsson et al.,Science, 237:1171-1176). Leukotrienes mediate a variety of signals ininflammation and immunity, and lipoxins serve as intra- andintercellular messengers in a variety of functions of vasculature andinflammation (Serhan et al, J. Bioenerg. Biomembr. 23:105-122, 1991.)Several flavonoids inhibit animal 5-lipoxygenase (Laughton et al.,Biochem. Pharmacol. 42:1673-1681). The inhibition of soybeanlipoxygenase activity by select cocoa polyphenol extracts was tested inthis work, and it was shown that cocoa polyphenol extract sub-fractionsinhibit lipoxygenase activity in vitro. Therefore the cocoa extractshave a utility in the prevention of atherosclerosis via the inhibitionof lipid oxidation by lipoxygenase.

[0070] It has been surprisingly found that the cocoa procyanidins haveutility in the treatment of diseases associated with COX/LOX. In Example12, COX was inhibited by individual cocoa procyanidins at concentrationssimilar to the known NSAID Indomethacin.

[0071] For COX inhibition, the preferred cocoa procyanidins areoligomers, where n is 2 to 18. In a preferred embodiment, the cocoaprocyanidins are oligomers where n is 2 to 10, more preferably 2 to 5.Examples of compounds eliciting inhibitory activity with respect toCOX/LOX include dimers, trimers, tetramers and pentamers.

[0072] Hence, given the significant inhibitory potency of the cocoaprocyanidins on COX-2, coupled with the cytotoxic effects on a putativeCOX-2 expression colon cancer cell line, the cocoa procyanid ins shouldpossess apoptotic activity as inhibitors of the multi step progressionleading to carcinomas, as well as activity as members of the NSAIDfamily of medications possessing a broad spectrum of prophylacticactivities.

[0073] Further, prostaglandins, the penultimate products of the COXcatalyzed conversion of arachidonic acid to prostaglandin H₂, areinvolved in inflammation, pain, fever, fetal development, labor andplatelet aggregation. Therefore, the cocoa procyanidins are efficaciousfor the same conditions as NSAIDs, e.g., against vascular disease, andstroke, etc. Indeed, the inhibition of platelet COX-1, which reducesthromboxane A₂ production, is the basis for the prophylactic effects ofaspirin on vascular disease.

[0074] Inflammation is the response of living tissues to injury. Itinvolves a complex series of enzyme activation, mediator release,extravasation of fluid, cell migration, tissue breakdown and repair.Inflammation is activated by phospholipase A₂, which liberatesarachidonic acid, the substrate for COX and LOX enzymes. COX convertsarachidonic acid to the prostaglandin PGE₂, the major eicosanoiddetected in inflammatory conditions ranging from acute edema to chronicarthritis. Its inhibition by NSAIDs is a mainstay for treatment.

[0075] Arthritis is one of the rheumatic diseases which encompass a widerange of diseases and pathological processes, most of which affect jointtissue. The basic structure affected by these diseases is the connectivetissue which includes synovial membranes, cartilage, bone, tendons,ligaments, and interstitial tissues. Temporary connective tissuesyndromes include sprains and strains, tendonitis, and tendon sheathabnormalities. The most serious forms of arthritis are rheumatoidarthritis, osteoarthritis, gout and systemic lupus erythematosus.

[0076] In addition to the rheumatic diseases, other diseases arecharacterized by inflammation. Gingivitis and periodontitis follows apathological picture resembling rheumatoid arthritis. Inflammatory boweldisease refers to idiopathic chronic inflammatory conditions of theintestine, ulcerative colitis and Crohn's disease. Spondylitis refers tochronic inflammation of the joints of the spine. There is also a highincidence of osteoarthritis associated with obesity.

[0077] Thus, the cocoa procyanidins have utility in the treatment ofconditions involving inflammation, pain, fever, and plateletaggregation.

[0078] The prostanoids and endothelins participate not only in animaldevelopment (e.g. nerve crest-derived structures) but also in theregulation of the cardiorespiratory systems of adult organisms (Hugginset al., ‘ET-1 Induction Of Cyclo-oxygenase-2 Expression In Rat MesangialCells’ 1993; Harbome, J. B., The Flavonoids: Advances in Research since1986′, Chapman and Hall, London, 1994; Prins et al., ‘Prostglandin E2and Prostacyclin Inhibit the Production of Endothelin from CulturedEndothelial Cells, J. Biol. Chem., 269:11938-11944, 1994). Vasculareffects of prostacyclin include decreased vessel contraction, plateletaggregation, thrombosis formation, smooth muscle cell proliferation, andthe entry of low-density lipoproteins into the arterial wall, while theendothelins (ET-1, ET-2, ET-3), PGE₂, and thromboxane inducevasoconstriction and platelet aggregation (Kuwaki et al., ‘Physiologicalrole of Brain ET on the Central Autonomic Control: From Neuron toKnockout Mouse’, 1997; Luscher T. F. ‘Platelet-vessel Wall Interactions:Role of Nitric Oxide, Prostglandins, and ET's. Balliere's Clin.Haemotol. 6:609-627, 1993). It has been shown by the inventors that, inan endothelial cell monolayer culture system, cocoa procyanidin extractsinduce prostacyclin cell release and inhibit endothelin cell release,hence promoting a state of vessel relaxation and decreased plateletaggregation. The claimed compounds therefore have utility asvasoprotectors in the treatment of vascular disease.

[0079] The inhibition of COX by the cocoa procyanidins would alsoinhibit the formation of postaglandins, e.g., PGD₂, PGE₂. Thus, thecocoa procyanidins have utility in the treatment of conditionsassociated with prostaglandins PGD₂ and PGE₂.

[0080] NO-Associated Utilities

[0081] Nitric oxide (NO) is known to inhibit platelet aggregation,monocyte adhesion and chemotaxis, and proliferation of vascular smoothmuscle tissue which are critically involved in the process ofatherogenesis. Evidence supports the view that NO is reduced inatherosclerotic tissues due to its reaction with oxygen free radicals.The loss of NO due to these reactions leads to increased platelet andinflammatory cell adhesion to vessel walls to further impair NOmechanisms of relaxation. In this manner, the loss of NO promotesatherogenic processes, leading to progressive disease states.

[0082] Hypertension is a leading cause of vascular diseases, includingstroke, heart attack, heart failure, irregular heart beat and kidneyfailure. Hypertension is a condition where the pressure of blood withinthe blood vessels is higher than normal as it circulates through thebody. When the systolic pressure exceeds 150 mm Hg or the diastolicpressure exceeds 90 mm Hg for a sustained period of time, damage is doneto the body. For example, excessive systolic pressure can rupture bloodvessels anywhere. When it occurs within the brain, a stroke results. Itcan also cause thickening and narrowing of the blood vessels which canlead to atherosclerosis. Elevated blood pressure can also force theheart muscle to enlarge as it works harder to overcome the elevatedresting (diastolic) pressure when blood is expelled. This enlargementcan eventually produce irregular heart beats or heart failure.Hypertension is called the “silent killer” because it causes no symptomsand can only be detected when blood pressure is checked.

[0083] The regulation of blood pressure is a complex event where onemechanism involves the expression of constitutive Ca⁺²/calmodulindependent form of nitric oxide synthase (NOS), abbreviated eNOS. NOproduced by this enzyme produces muscle relaxation in the vessel(dilation), which lowers the blood pressure. When the normal level of NOproduced by eNOS is not produced, either because production is blockedby an inhibitor or in pathological states, such as atherosclerosis, thevascular muscles do not relax to the appropriate degree. The resultingvasoconstriction increases blood pressure and may be responsible forsome forms of hypertension.

[0084] Vascular endothelial cells contain eNOS. NO synthesized by eNOSdiffuses in diverse directions, and when it reaches the underlyingvascular smooth muscle, NO binds to the heme group of guanylyl cyclase,causing an increase in cGMP. Increased cGMP causes a decrease inintracellular free Ca⁺². Cyclic GMP may activate a protein kinase thatphosphorylates Ca⁺² transporters, causing Ca⁺² to be sequestered inintracellular structures in the muscle cells. Since muscle contractionrequires Ca⁺², the force of the contraction is reduced as the Ca⁺²concentration declines. Muscle relaxation allows the vessel to dilate,which lowers the blood pressure. Inhibition of eNOS therefore causesblood pressure to increase.

[0085] When the normal level of NO is not produced, either becauseproduction is blocked by administration of an NOS inhibitor or possibly,in pathological states, such as atherosclerosis, the vascular muscles donot relax to the appropriate degree. The resulting vasoconstrictionincreases blood pressure and may be responsible for some forms ofhypertension. There is considerable interest in finding therapeutic waysto increase the activity of eNOS in hypertensive patients, but practicaltherapies have not been reported. Pharmacological agents capable ofreleasing NO, such as nitroglycerin or isosorbide dinitrate, remainmainstays of vasorelaxant therapy.

[0086] Although the cocoa procyanidins inhibit the oxidation of LDL, themore comprehensive effects of these compounds is their multidimensionaleffects on atherosclerosis via NO. NO modulation by the cocoaprocyanidins brings about a collage of beneficial effects, including themodulation of hypertension, lowering NO affected hypercholesterolemia,inhibiting platelet aggregation and monocyte adhesion, all of which areinvolved with the progression of atherosclerosis.

[0087] The role of NO in the immune system is different from itsfunction in blood vessels. Macrophages contain a form of NOS that isinducible, rather than constitutive, referred to as iNOS. Transcriptionof the iNOS gene is controlled both positively and negatively by anumber of biological response modifiers called cytokines. The mostimportant inducers are gamma-interferon, tumor necrosis factor,interleukin-1, interleukin-2 and lipopolysaccharide (LPS), which is acomponent of the cell walls of gram negative bacteria. Stimulatedmacrophages produce enough NO to inhibit ribonuclease reductase, theenzyme that converts ribonucleotides to the deoxyribonucleotidesnecessary for DNA synthesis. Inhibition of DNA synthesis may be animportant way in which macrophages and other tissues possessing iNOS caninhibit the growth of rapidly dividing tumor cells or infectiousbacteria.

[0088] With regard to the effects of NO and infectious bacteria,microorganisms play a significant role in infectious processes whichreflect body contact and injury, habits, profession, environment of theindividual, as well as food bome diseases brought about by improperstorage, handling and contamination.

[0089] The cocoa procyanidins, combinations thereof, and compositionscontaining them are useful in the treatment of conditions associatedwith modulating NO concentrations.

[0090] Inhibition of Platelet Aggregation

[0091] Blood platelets play a major role in coronary artery disease.Platelets are found at the site of early atherosclerotic lesions. Whenactivated, they secrete potent mitogenic factors such as plateletderived growth factor, transforming growth factor-α and epidermal growthfactor, which lead to smooth muscle proliferation and progression ofatherosclerotic lesions.

[0092] Additionally, enhanced platelet reactivity and spontaneousplatelet aggregates are crucially involved in thrombus formation, whichis largely responsible for the pathogenesis of acute myocardialinfarction, unstable angina and percutaneous coronary intervention.Therapy with antiplatelet agents (such as aspirin) significantlydecrease the incidence of primary and secondary coronary events(Schafer, A. I. “Antiplatelet Therapy”, Am. J. Med. 101:199-209, 1996).

[0093] Platelet function depends on the interactions of membraneglycoproteins, such as GPIIb/IIIa, which act as receptors for adhesiveproteins on the platelet surface. Agonists of GPIIb/IIIa facilitate theconformational change necessary for the receptor to become receptive toligands which bind simultaneously to two separate platelets, therebycross-linking and aggregating the platelets. Antagonists of theGPIIb/IIIa receptor prevent activation of the receptor, therebypreventing platelet activation. Pharmacological intervention directedagainst the GPIIb/IIIa receptor is therefore being pioneered in thetreatment of ischemic heart disease. Several GPIIb/IIIa agonists havebeen used in clinical trials in recent years, and have been shown tohave considerable benefit in various treatment regimes (Vorchheimer etal, JAMA, 281:15:1407-1413, 1999).

[0094] It has been found that consumption of a cocoa beverage with anenhanced cocoa procyanidin content results in the suppression ofactivation of the GPIIbIIIa receptor. Therefore, the cocoa procyanidinshave a utility in the treatment and prevention of atherosclerosis.

[0095] It has been shown that the cocoa procyanidins have a potentantioxidant activity (as disclosed in Romanczyk et al, US Ser. No.5,554,645), which is known to be due to the inhibition of free radicals.Given that NO is a free radical and that the cocoa procyanidins arestrong antioxidants, it was suspected that the administration of thecocoa procyanidins to experimental in vitro and in vivo models wouldhave caused a reduction in NO levels. Any reduction in NO would haveresulted in a hypertensive, rather than a hypotensive effect. Contraryto expectations, the cocoa procyanidins elicited increases in NO in thein vitro experiments and produced a hypotensive effect in the in vivostudies (Examples 17 and 18). These results were not anticipated andcompletely unexpected.

[0096] Example 8 describes the hypotensive effects elicited by the cocoaprocyanidins in an in vivo animal model, thus demonstrating the efficacyof the cocoa procyanidins in the treatment of hypertension. In thisexample, the cocoa procyanidins, combinations thereof and compositionscomprising the same comprise oligomers wherein n is 2 to 18, andpreferably, n is 2 to 10.

[0097] Example 9 describes the modulation of NO production by the cocoaprocyanidins in an in vitro model. In this example, the cocoaprocyanidins, combinations thereof and compositions comprising the samecomprise oligomers wherein n is 2 to 18, and preferably n is 2 to 10.

[0098] Further, Example 6 provides evidence for the formation of Cu⁺²-,Fe⁺²- and Fe⁺³-oligomer complexes detected by MALDI/TOF/MS. Theseresults indicate that the cocoa procyanidins can complex with copperand/or iron ions to minimize their effects on LDL oxidation.

[0099] Example 10 describes the effects of the cocoa procyanidins onmacrophage NO production. In this example, the results demonstrate thatthe cocoa procyanidins induce monocyte/macrophage NO production, bothindependent and dependent of stimulation by lipopolysaccharide (LPS) orcytokines. Macrophages producing NO can inhibit the growth of infectiousbacteria.

[0100] Formulations and Methods

[0101] Therefore, collectively, the cocoa procyanidins, combinationsthereof and compositions containing them have exhibited a wide array ofactivities and functions including NO or NO-synthase modulator,non-steroidal anti-inflammatory agent, platelet activation modulator,nonsteroidal anti-inflammatory agent, modulator of the immune system,and cyclo-oxygenase and/or lipoxygenase modulator.

[0102] For treatment or prevention of vascular diseases includingrestenosis and atherosclerosis, a cocoa procyanidin or mixture of cocoaprocyanidin monomer and/or oligomers or a composition comprising cocoaprocyanidin or procyanidins, alone or with other treatment, may beadministered as desired by the skilled medical practitioner, from thisdisclosure and knowledge in the art, e.g., at the first signs orsymptoms of restenosis and/or atherosclerosis, immediately prior to,concomitant with or after angioplasty, or as soon thereafter as desiredby the skilled medical practitioner, without any undue experimentationrequired; and the administration of the inventive compound or compoundsor a composition thereof, alone or with other treatment, may becontinued as a regimen, e.g., monthly, bimonthly, biannually, annually,or in some other regimen, by the skilled medical practitioner for suchtime as is necessary, without any undue experimentation required.

[0103] Further, the cocoa procyanidins, combinations thereof andcompositions comprising the same have been shown to produce ahypotensive effect in vivo and induce NO in vitro. These results havepractical application in the treatment of hypertension and in clinicalsituations involving hypercholesterolemia, where NO levels are markedlyreduced.

[0104] Formulations of the cocoa procyanidins, combinations thereof andcompositions containing them can be prepared with standard techniqueswell known to those skilled in the pharmaceutical, food science, medicaland veterinary arts, in the form of a liquid, suspension, tablet,capsule, injectable solution or suppository, for immediate orslow-release of the active compounds. The carrier may also be apolymeric delayed release system. Synthetic polymers are particularlyuseful in the formulation of a composition having controlled release. Anearly example of this was the polymerization of methyl methacrylate intospheres having diameters less than one micron to form so-called nanoparticles, reported by Kreuter, J., Microcapsules and Nanoparticles inMedicine and Pharmacology, M. Donbrow (Ed). CRC Press, p. 125-148.

[0105] A frequent choice of a carrier for pharmaceuticals and morerecently for antigens is poly (d, 1-lactide-co-glycolide) (PLGA). Thisis a biodegradable polyester that has a long history of medical use inerodable sutures, bone plates and other temporary prostheses where ithas not exhibited any toxicity. A wide variety of pharmaceuticals havebeen formulated into PLGA microcapsules. A body of data has accumulatedon the adaption of PLGA for controlled release, for example, as reviewedby Eldridge, J. H., et al. Current Topics in Microbiology andImmunology, 1989, 146:59-66. The entrapment in PLGA microspheres of 1 to10 microns in diameter can be effective when administered orally. ThePLGA microencapsulation process uses a phase separation of awater-in-oil emulsion. The cocoa procyanidins are prepared as an aqueoussolution and the PLGA is dissolved in a suitable organic solvents suchas methylene chloride and ethyl acetate. These two immiscible solutionsare co-emulsified by high-speed stirring. A non-solvent for the polymeris then added, causing precipitation of the polymer around the aqueousdroplets to form embryonic microcapsules. The microcapsules arecollected, and stabilized with one of an assortment of agents includingpolyvinyl alcohol (PVA), alginates and methyl cellulose. The solvent issubsequently removed by either drying in vacuo or solvent extraction.

[0106] Additionally, with regard to the preparation of slow-releaseformulations, the disclosures of U.S. Pat. Nos. 5,024,843, 5,091,190,5,082,668, 4,612,008 and 4,327,725 are hereby incorporated herein byreference.

[0107] Additionally, selective processing coupled with theidentification of cocoa genotypes of interest could be used to prepareStandard-of-Identity (SOI) and non-SOI chocolate products as vehicles todeliver the active compounds to a patient in need of treatment for thedisease conditions described above, as well as a means for the deliveryof conserved levels of the cocoa procyanidins.

[0108] A method of producing cocoa butter and/or cocoa solids havingconserved levels of cocoa polyphenols from cocoa beans uses a uniquecombination of processing steps which does not require separate beanroasting or liquor milling equipment, allowing for the option ofprocessing cocoa beans without exposure to severe thermal treatment forextended periods of time and/or the use of solvent extraction of fat.The benefit of this process lies in the enhanced conservation ofpolyphenols in contrast to that found in traditional cocoa processing,such that the ratio of the initial amount of polyphenol found in theunprocessed bean to that obtainable after processing is less than orequal to 2. Partially defatted cocoa solids having a high cocoapolyphenol (CP) content, including a high cocoa procyanidin content, canbe obtained by processing the cocoa beans directly to cocoa solidswithout a bean or nib roasting step. This method conserves the cocoapolyphenols because it omits the traditional roasting step. This methodconsists essentially of the steps of: (a) heating the cocoa beans to aninternal bean temperature just sufficient to reduce the moisture contentto about 3% by weight and to loosen the cocoa shell; (b) winnowing thecocoa nibs from the cocoa shells; (c) screw pressing the cocoa nibs; and(d) recovering the cocoa butter and partially defatted cocoa solidswhich contain cocoa polyphenols including cocoa procyanidins.Optionally, the cocoa beans are cleaned prior to the heating step, e.g.,in an air fluidized bed density separator. The winnowing can also becarried out in the air fluidized bed density separator. Preferably, thecocoa beans are heated to an internal bean temperature of about 100° C.to about 110° C., more preferably less than about 105° C., typicallyusing a infra red heating apparatus for about 3 to 4 minutes. Ifdesired, the cocoa solids can be alkalized and/or milled to a cocoapowder.

[0109] The internal bean temperature (IBT) can be measured by filling aninsulated container such as a thermos bottle with beans (approximately80-100 beans). The insulated container is then appropriately sealed inorder to maintain the temperature of the sample therein. A thermometeris inserted into the bean-filled insulated container and the temperatureof the thermometer is equilibrated with respect to the beans in thethermos. The temperature reading is the IBT temperature of the beans.IBT can also be considered the equilibrium mass temperature of thebeans.

[0110] Cocoa beans can be divided into four categories based on theircolor: predominately brown (fully fermented), purple/brown, purple, andslaty (unfermented). Preferably, the cocoa solids are prepared fromunderfermented cocoa beans which have a higher cocoa polyphenol contentthan fermented beans. Underfermented beans include slaty cocoa beans,purple cocoa beans, mixtures of slaty and purple cocoa beans, mixturesof purple and brown cocoa beans, or mixture of slaty, purple, and browncocoa beans. More preferably, the cocoa beans are slaty and/or purplecocoa beans.

[0111] As discussed above, the cocoa polyphenol (CP) content, includingthe cocoa procyanidin content, of roasted cocoa nibs, chocolate liquor,and partially defatted or nonfat cocoa solids is higher when they areprepared from cocoa beans or blends thereof which are underfermented,i.e., beans having a fermentation factor of 275 or less.

[0112] The “fermentation factor” is determined using a grading systemfor characterizing the fermentation of the cocoa beans. Slaty isdesignated 1, purple is 2, purple/brown is 3, and brown is 4. Thepercentage of beans falling within each category is multiplied by theweighted number. Thus, the “fermentation factor” for a sample of 100%brown beans would be 100×4 or 400, whereas for a 100% sample of purplebeans it would be 100×2 or 200. A sample of 50% slaty beans and 50%purple beans would have a fermentation factor of 150 [(50×1)+(50×2)].

[0113] An extract containing cocoa polyphenols including cocoaprocyanidins can be prepared by solvent extracting the partiallydefatted cocoa solids, and purifying the extract to remove the xanthinescaffeine and theobromine.

[0114] Such compositions can be administered to a subject or patient inneed of such administration in dosages and by techniques well known tothose skilled in the medical, nutritional or veterinary arts taking intoconsideration the data herein, and such factors as the age, sex, weight,genetics and condition of the particular subject or patient, the routeof administration, relative concentration of particular oligomers, andtoxicity (e.g., LD₅₀).

[0115] Suitable compositions of the invention for human or veterinaryuse include edible compositions for oral administration, such solid orliquid formulations, for example, capsules, tablets, pills and the like;chewable solid formulations, beverage formulations, or dried beverageformulations for reconstitution; liquid preparations for orificeadministration, e.g., by oral, by nasal, by anal, by vaginaladministration via suspensions, syrups or elixirs; and ingestablepreparations for parenteral, subcutaneous, intradermal, intramuscular orintravenous administration e.g., suspensions or emulsions. The abovecompositions maybe chocolate flavored if the high cocoa polyphenolsolids are used in the composition. However, if the cocoa extract isused in the composition, chocolate or other flavoring agents may beincluded in the composition, particularly if the composition is anedible composition. The active ingredient in the compositions maycomplex with proteins and, when administered into the bloodstream,clotting may occur due to the precipitation of blood proteins. Theskilled artisan should take this into account. In such compositions theactive cocoa procyanidin may be in admixture with a suitable carrier,diluent, or excipient such as sterile water, physiological saline,glucose, DMSO, ethanol, or the like. The cocoa extract or cocoaprocyanidin fractions can be provided in lyophilized form forreconstituting for example, in an edible liquid or in isotonic aqueous,saline, glucose or DMSO buffer. In certain saline solutions, someprecipitation has been observed. Precipitation may be employed as ameans to isolate cocoa procyanidins, e.g. by a “salting out” procedure.

[0116] Example 14 describes the utility of a procyanidin-enriched cocoabeverage in the inhibition of platelet activation. A preferred beverageor beverage mix comprises: high cocoa polyphenol solids and/or cocoaextract; and optionally a natural or artificial sweetener, a natural orsynthetic flavorant, and a dairy product. The beverage may also be acarbonated beverage. The sweetener may be a sugar syrup/solids, or asugar substitute. The term “sugar substitute” includes bulking agents,sugar alcohols, (i.e. polyols such as glycerol), high potency sweetenersor combinations thereof. Nutritive carbohydrate sweeteners with varyingdegrees of sweetness intensity may be any of those typically used in theart and include, but are not limited to, sucrose, dextrose, fructose,lactose, maltose, glucose syrup solids, corn syrup solids, invert sugar,hydrolyzed lactose, honey, maple sugar, brown sugar, molasses and thelike. Sugar substitutes may partially or totally replace the nutritivecarbohydrate sweetener. High potency sugar substitutes includeaspartame, cyclamates, saccharin, acesulfame-K, neohesperidin,dihydrochalcone, sucralose, alitame, stevia sweeteners, glycyrrhizin,thaumatin and the like as well as mixtures thereof. Exemplary sugaralcohols include those typically used in the art such as sorbitol,mannitol, xylitol, maltitol, isomalt, lactitol and the like. Exemplarydairy components are non-fat milk solids, milk fat, sweet cream,buttermilk and skim milk.

[0117] Example 15 describes the formulation of cocoa procyanidinstablets, for use in the pharmaceutical, diet supplement and food areas.Example 16 describes the preparation of the cocoa procyanidins ascapsules for similar applications. Example 17 describes the preparationof Standard of Identity (SOI) and non-SOI chocolates containing the highcocoa polyphenol extract or cocoa solids obtained from methods describedherein.

[0118] Kits

[0119] The active cocoa extract may be provided in a kit includes aseparate container containing a suitable carrier, diluent or excipient,and optionally other active ingredients which will depend upon thehealth benefit to be achieved, and additional agent(s) which can beprovided in separate container(s) or in admixture with the active cocoaprocyanidin(s). The kit may also include instructions for mixing orcombining the ingredients and/or the administration.

EXAMPLES Example 1 Cocoa Source and Method of Preparation

[0120] Several Theobroma cacao genotypes which represent the threerecognized horticultural races of cocoa (Enriquez et al, Cocoa CultivarsRegister IICA, Turrialba, Cost Rica 1967; Engels, Genetic Resources ofCacao: A Catalogue of the CATIE Collection, Tech. Bull. 7, Turrialba,Costa Rica, 1981) were obtained from the three major cocoa producingorigins of the world. A list of those genotypes used in this study areshown in Table 1. Harvested cocoa pods were opened and the beans withpulp were removed for freeze drying. The pulp was manually removed fromthe freeze dried mass and the beans were subjected to analysis asfollows. The unfermented, freeze dried cocoa beans were first manuallydehulled, and ground to a fine powdery mass with a TEKMAR Mill. Theresultant mass was then defatted overnight by Soxhlet extraction usingredistilled hexane as the solvent. Residual solvent was removed from thedefatted mass by vacuum at ambient temperature. TABLE 1 Description ofTheobroma cacao Source Material GENOTYPE ORIGIN HORTICULTURAL RACE UIT-1Malaysia Trinitario Unknown West Africa Forastero ICS-100 BrazilTrinitario (Nicaraguan Criollo ancestor) ICS-39 Brazil Trinitario(Nicaraguan Criollo ancestor) UF-613 Brazil Trinitario EEG-48 BrazilForastero UF-12 Brazil Trinitario NA-33 Brazil Forastero

Example 2 Procyanidin Extraction Procedures

[0121] A. Method 1

[0122] Procyanidins were extracted from the defatted, unfermented,freeze dried cocoa beans of Example 1 using a modification of the methoddescribed by Jalal and Collin (‘Polyphenols of Mature Plant, Seedlingand Tissue Cultures of Theobroma Cacoa’, Phytochemistry, 6, 1377-1380,1977). Procyanidins were extracted from 50 gram batches of the defattedcocoa mass with 2×400 mL 70% acetone/deionized water followed by 400 mL70% methanol/deionized water. The extracts were pooled and the solventsremoved by evaporation at 45° C. with a rotary evaporator held underpartial vacuum. The resultant aqueous phase was diluted to 1 L withdeionized water and extracted 2× with 400 mL CHCl₃. The solvent phasewas discarded. The aqueous phase was then extracted 4× with 500 mL ethylacetate. Any resultant emulsions were broken by centrifugation on aSorvall RC 28S centrifuge operated at 2,000×for 30 min. at 10° C. To thecombined ethyl acetate extracts, 100-200 nL deionized water was added.The solvent was removed by evaporation at 45° C. with a rotaryevaporator held under partial vacuum. The resultant aqueous phase wasfrozen in liquid N₂ followed by freeze drying on a LABCONCO Freeze DrySystem. The yields of crude procyanidins that were obtained from thedifferent cocoa genotypes are listed in Table 2. TABLE 2 CrudeProcyanidin Yields HORTICULTUREAL GENOTYPE ORIGIN RACE UIT-1 Malaysia3.81 Unknown West Africa 2.55 ICS-100 Brazil 3.42 ICS-39 Brazil 3.45UF-613 Brazil 2.98 EEG-48 Brazil 3.15 UF-12 Brazil 1.21 NA-33 Brazil2.23

[0123] B. Method 2

[0124] Alternatively, procyanidins are extracted from defatted,unfermented, freeze dried cocoa beans of Example 1 with 70% aqueousacetone. Ten grams of defatted material was slurried with 100 mL solventfor 5-10 min. The slurry was centrifuged for 15 min. at 4° C. at 3000×gand the supernatant passed through glass wool. The filtrate wassubjected to distillation under partial vacuum and the resultant aqueousphase frozen in liquid N₂, followed by freeze drying on a LABCONCOFreeze Dry System. The yields of crude procyanidins ranged from 15-20%.

[0125] Without wishing to be bound by any particular theory, it isbelieved that the differences in crude yields reflected variationsencountered with different genotypes, geographical origin, horticulturalrace, and method of preparation.

Example 3 Partial Purification of Cocoa Procyanidins by Gel PermeationChromatography

[0126] A. Method 1

[0127] Procyanidins obtained from Example 2 were partially purified byliquid chromatography on Sephadex LH-20 (28×2.5 cm). Separations wereaided by a step gradient from deionized water into methanol. The initialgradient composition started with 15% methanol in deionized water whichwas followed step wise every 30 min. with 25% methanol in deionizedwater, 35% methanol in deionized water, 70% methanol in deionized water,and finally 100% methanol. The effluent following the elution of thexanthine alkaloids (caffeine and theobromine) was collected as a singlefraction. The fraction yielded a xanthine alkaloid free subfractionwhich was submitted to further subfractionation to yield fivesubfractions designated MM2A through MM2E. The solvent was removed fromeach subfraction by evaporation at 45° C. with a rotary evaporator heldunder partial vacuum. The resultant aqueous phase was frozen in liquidN₂ and freeze dried overnight on a LABCONCO Freeze Dry System. Arepresentative gel permeation chromatogram showing the fractionation isshown in FIG. 3. Approximately, 100 mg of material was subfractionatedin this manner.

[0128] Chromatographic Conditions: Column; 28×2.5 cm Sephadex LH-20,Mobile Phase: Methanol/Water Step Gradient, 15:85, 25:75, 35:65, 70:30,100:0 Stepped at ½ Hour Intervals, Flow Rate; 1.5 mL/min, Detector; UVat λ₁=254 nm and λ₂=365 nm, Chart Speed: 0.5 mm/min, Column Load; 120mg.

[0129] B. Method 2

[0130] Procyanidins obtained as in Example 2 were partially purified byliquid chromatography on Sephadex LH 20 (72.5×2.5 cm), using 100%methanol as the eluting solvent, at a flow rate of 3.5 mL/min. Fractionsof the eluent were collected after the first 1.5 hours, and thefractions were concentrated by a rotary evaporator, redissolved in waterand freeze dried. These fractions were referred to as pentamer enrichedfractions. Approximately 2.00 g of the extract obtained from Example 2was subfractionated in this manner.

Example 4 Analytical HPLC Analysis of Procyanidin Extracts

[0131] Method 1. Reverse Phase Separation

[0132] Procyanidin extracts obtained from Examples 2 & 3 were filteredthrough a 0.45μ filter and analyzed by a Hewlett Packard 1090 ternaryHPLC system equipped with a Diode Array detector and a HP model 1046AProgrammable Fluorescence Detector. Separations were effected at 45° C.on a Hewlett-Packard 5μ Hypersil ODS column (200×2.1 mm). The flavanolsand procyanidins were eluted with a linear gradient of 60% B into Afollowed by a column wash with B at a flow rate of 0.3 mL/min. Themobile phase composition was B=0.5% acetic acid in methanol and A=0.5%acetic acid in nanopure water. Acetic acid levels in A and B mobilephases can be increased to 2%. Components were detected by fluorescence,where λ_(ex)=276 nm and λ_(ex)=316 nm and by UV at 280 nm.Concentrations of (+)-catechin and (−)-epicatechin were determinedrelative to reference standard solutions. Procyanidin levels wereestimated by using the response factor for (−)-epicatechin. Arepresentative HPLC chromatogram showing the separation of the variouscomponents is shown in FIG. 4 for one cocoa genotype. Similar HPLCprofiles were obtained from the other cocoa genotypes.

[0133] HPLC Conditions: Column: 200 × 2.1 mm Hewlett Packard HypersilODS (5 μ) Guard column:  20 × 2.1 mm Hewlett Packard Hypersil ODS (5 μ)Detectors: Diode Array @ 280 nm Fluorescence λex = 276 nm; λem = 316 nm.Flow rate: 0.3 mL/min. Column 45° C. Temperature:

[0134] Gradient: 0.5% Acetic Acid in 0.5% Acetic acid in Time (min)nanopure water methanol 0 100 0 50 40 60 60 0 100

[0135] Method 2. Normal Phase Separation

[0136] Procyanidin extracts obtained from previous examples werefiltered through a 0.45μ filter and analyzed by a Hewlett Packard 1090Series II HPLC system equipped with a HP model 1046A ProgrammableFluorescence detector and Diode Array detector. Separations wereeffected at 37° C. on a 5μ Phenomenex Lichrosphere® Silica 100 column(250×3.2 mm) connected to a Supelco Supelguard LC—Si 5μ guard column(20×4.6 mm). Procyanidins were eluted by linear gradient under thefollowing conditions: (Time, % A, % B); (0, 82, 14), (30, 67.6, 28.4),(60, 46, 50), (65, 10, 86), (70, 10, 86) followed by an 8 min.re-equilibration. Mobile phase composition was A=dichloromethane,B=methanol, and C=acetic acid:water at a volume ratio of 1:1. A flowrate of 0.5 mL/min. was used. Components were detected by fluorescence,where λ^(ex)=276 nm and λ^(em)=316 nm or by UV at 280 nm. Arepresentative HPLC chromatogram showing the separation of the variousprocyanidins is shown in FIG. 5 for one genotype. Similar HPLC profileswere obtained from other cocoa genotypes.

[0137] HPLC Conditions: 250×3.2 mm Phenomenex Lichrosphere® Silica 100column (5μ) 20×4.6 mm Supelco Supelguard LC—Si (5μ) guard columnDetectors: Photodiode Array @ 280 nm Fluorescence λ^(ex) = 276 nm;λ^(em) = 316 nm. Flow rate: 0.5 mL/min. Column Temperature: 37° C.

[0138] Gradient: Acetic Acid/Water Time (min.) CH₂—CI₂ Methanol (1:1) 082 14 4 30 67.6 28.4 4 60 46 50 4 65 10 86 4 70 10 86 4

Example 5 Purification of Oligomeric Fractions from Pentamer EnrichedFractions

[0139] Preparative Normal Phase Separation

[0140] The pentamer enriched fractions obtained as in Example 4 werefurther purified by preparative normal phase chromatography by modifyingthe method of Rigaud et al., (J. Chrom. 654: 255-260, 1993).

[0141] Separations were affected at ambient temperature on a 5μSupelcosil LC—Si 100 Å column (50×2 cm), with an appropriate guardcolumn. Procyanidins were eluted by a linear gradient under thefollowing conditions: (time, % A, % B, flow rate); (0, 92.5, 7.5, 10);(10, 92.5, 7.5, 40); (30, 91.5, 18.5, 40); (145, 88, 22, 40); (150, 24,86, 40); (155, 24, 86, 50); (180, 0, 100, 50). Prior to use, the mobilephase components were mixed by the following protocol:

[0142] Solvent A preparation (82% CH₂Cl₂, 14% methanol, 2% acetic acid,2% water):

[0143] 1. Measure 80 mL of water and dispense into a 4 L bottle.

[0144] 2. Measure 80 mL of acetic acid and dispense into the same 4 Lbottle.

[0145] 3. Measure 560 mL of methanol and dispense into the same 4 Lbottle.

[0146] 4. Measure 3280 mL of methylene chloride and dispense into the 4L bottle.

[0147] 5. Cap the bottle and mix well.

[0148] 6. Purge the mixture with high purity Helium for 5-10 minutes todegas.

[0149] Repeat steps 1-6 two times to yield 8 volumes of solvent A.

[0150] Solvent B preparation (96% methanol, 2% acetic acid, 2% water):

[0151] 1. Measure 80 mL of water and dispense into a 4 L bottle.

[0152] 2. Measure 80 nL of acetic acid and dispense into the same 4 Lbottle.

[0153] 3. Measure 3840 nL of methanol and dispense 3840 mL of methanoland dispense into the same 4 L bottle.

[0154] 4. Cap the bottle and mix well.

[0155] 5. Purge the mixture with high purity Helium for 5-10 minutes todegas.

[0156] Repeat steps 1-5 to yield 4 volumes of solvent B. Mobile phasecomposition was A=methylene chloride with 2% acetic acid and 2% water;B=methanol with 2% acetic acid and 2% water. The column load was 0.7 gin 7 mL components were detected by UV at 254 nm. A typical preparativenormal phase HPLC separation of cocoa procyanidins is shown in FIG. 5.

[0157] HPLC Conditions: Column: 50×2 cm 5μ Supelcosil LC—Si run @ambient temperature.

[0158] Mobile Phase: A=Methylene Chloride with 2%

[0159] Acetic Acid and 2% Water.

[0160] B=Methanol with 2% Acetic Acid and 2% Water.

[0161] Gradient/Flow Profile: TIME FLOW RATE (MIN) % A % B (mL/min) 092.5 7.5 10 10 92.5 7.5 40 30 91.5 8.5 40 145 88.0 22.0 40 150 24.0 86.040 155 24.0 86.0 50 180 0.0 100.0 50

Example 6 MALDI-TOF/MS Analysis of High Molecular Weight ProcyanidinOligomers

[0162] An analytical examination was made on GPC eluants associated withhigh molecular weight procyanidin oligomers as prepared in Example 3,Method A. The objective was to determine whether procyanidin oligomerswith n>18 were present. If present, these oligomers represent additionalcocoa procyanidins. Adjustments to existing methods of isolation,separation and purification embodied in the invention can be made toobtain these oligomers for subsequent in vitro and in vivo evaluationfor anti-cancer, anti-tumor or antineoplastic activity, antioxidantactivity, inhibit DNA topoisomerase II enzyme, inhibit oxidative damageto DNA, and have antimicrobial, NO or NO-synthase, apoptosis, plateletaggregation, and blood or in vivo glucose-modulating activities, as wellas efficacy as non-steroidal anti-inflammatory agents.

[0163]FIG. 6 represents a MALDI-TOF mass spectrum of the GPC eluantsample described above. The [M+Na]⁺ and/or [M+K]⁺ and/or [M+2Na]⁺ ionscharacterizing procyanidin oligomers representative of tetramers throughoctadecamers are clearly evident.

[0164] It was learned that an acid and heat treatment will cause thehydrolysis of procyanidin oligomers. Therefore, the inventioncomprehends the controlled hydrolysis of high molecular weightprocyanidin oligomers (e.g. where n is 13 to 18) as a method to preparelower molecular weight procyanidin oligomers (e.g. where n is 2 to 12).TABLE 3 Composition of Fractions Obtained: goes with Example 3 DrimerOthers Fraction Monomer (% Trimer Tetramer Pentamer Hexamer HeptamerOctamer Nonamer Decamer Undecamer (% (Time) (% Area) Area) (% Area) (%Area) (% Area) (% Area) (% Area) (% Area) (% Area) (% Area) (% Area)Area) 1:15 3 8 16 3 ND ND ND ND ND ND ND ND 1.44 67 19 10 3 1 tr tr trtr tr tr tr 2:13 30 29 24 11 4 1 tr tr tr tr tr tr 2.42 2 16 31 28 15 62 tr tr tr tr tr 3:11 1 12 17 25 22 13 7 2 1 tr tr tr 3:40 tr 18 13 1820 15 10 5 2 tr tr tr 4:09 tr 6 8 17 21 19 14 8 4 2 tr tr

[0165] TABLE 4 Theobroma and Herrania Species Procyanidin Levels ppm(μg/g) in defatted powder Oligomer Mon- Tetra- Penta- Hexa- SAMPLE omerDimer Trimer mer mer mer Heptamer Octamer Nonamer Decamer UndecamerTotal T. grandiflorum x 3822 3442 5384 4074 3146 2080 850 421 348 198tr⁺ 23,765 T. obovaum 1¹ T. grandiflorum x 3003 4098 5411 3983 3931 19141090 577 356 198 tr 23,561 T. obovaum 2¹ T. grandiflorum x 4990 49807556 5341 4008 2576 1075 598 301 144 tr 31,569 T. obovaum 3A¹ T.grandiflorum x 3880 4498 6488 4930 3706 2560 1208 593 323 174 tr 28,360T. obovaum 3B¹ T. grandiflorum x 2647 3591 5328 4240 3304 2380 1506 815506 249 tr 24,566 T. obovaum 4¹ T. grandiflorum x 2754 3855 5299 38722994 1990 1158 629 356 196 88 23,194 T. obovaum 6¹ T. grandiflorum x3212 4134 7608 4736 3590 2274 936 446 278 126 ND* 23,750 T. oboratumSIN¹ T. obovatum 1 ¹ 3662 5683 9512 5358 3858 2454 1207 640 302 144 ND32,820 T. grandiflorum 2608 2178 3090 2704 2241 1586 900 484 301 148 tr16,240 TEFFE² T. grandiflorum 4773 4096 5289 4748 3804 2444 998 737 335156 tr 27,380 TEFFE x T. grandiflorum ² T. grandiflorum x 4752 3336 49163900 3064 2039 782 435 380 228 ND 23,832 T. subincanum ¹ T. obovatum x3379 3802 5836 3940 2868 1807 814 427 271 136 tr 23,280 T. subincanum ¹T. speciosum x 902 346 1350 217 152 120 60 tr tr ND ND 3,147 T.sylvestris ¹ T. microcarpum ² 5694 3250 2766 1490 822 356 141 tr ND NDND 14,519 T. cacao, 21,929 10,072 10,106 7788 5311 3242 1311 626 422 146tr 60,753 SIAL 659, t0 T. cacao, 21,088 9762 9119 7094 4774 2906 1364608 361 176 tr 57,252 SIAL 659, t24 T. cacao, 20,887 9892 9474 7337 49062929 1334 692 412 302 tr 58,165 SIAL 659, t48 T. cacao, 9552 5780 50623360 2140 1160 464 254 138 tr ND 27,910 SIAL 659, t96 T. cacao, 85814665 4070 2527 1628 888 326 166 123 tr ND 22,974 SIAL 659, t120 Pod Rec.10/96, 869 1295 545 347 175 97 tr *ND ND 33329 Herrania mariae SampleRec. 130 354 151 131 116 51 tr ND ND 933 prior to 10/96, Herrania mariae

Example 7 Effect of Procvanidins on NO

[0166] Method A.

[0167] The purpose of this study was to establish the relationshipbetween procyanidins and NO, which is known to induce cerebral vasculardilation. The effects of monomers and higher oligomers, inconcentrations ranging from 100 μg/mL to 0.1 μg/mL, on the production ofnitrates (the catabolites of NO), from HUVEC (Human umbilical veinendothelial cells) is evaluated. HUVEC (from Clonetics) is investigatedin the presence or absence of each procyanidin for 24 to 48 hours. Atthe end of the experiments, the supernatants are collected and thenitrate content determined by calorimetric assay. In separateexperiments, HUVEC is incubated with acetylcholine, which is known toinduce NO production, in the presence or absence of procyanidins for 24to 48 hours. At the end of the experiments, the supernatants arecollected and nitrate content is determined by calorimetric assay. Therole of NO is ascertained by the addition of nitroarginine or(1)—N-methyl arginine, which are specific blockers of NO synthase.

[0168] Method B. Vasorelaxation of Phenylephrine-Induced Contracted RatArtery

[0169] The effects of each of the procyanidins (100 μg/mL to 0.11 g/mL)on the rat artery is the target for study of vasorelaxation ofphenylephrine-induced contracted rat artery. Isolated rat artery isincubated in the presence or absence of procyanidins and alteration ofthe muscular tone is assessed by visual inspection. Both contraction orrelaxation of the ray artery is determined. Then, using other organs,precontraction of the isolated rat artery is induced upon addition ofepinephrine. Once the contraction is stabilized, procyanidins are addedand contraction or relaxation of the rat artery is determined. The roleof NO is ascertained by the addition of nitroarginine or (1)—N-methylarginine. The acetylcholine-induced relaxation of NO-relatedphenylephrine-precontracted rat aorta is shown in FIG. 7.

[0170] Method C. Induction of Hypotension in the Rat

[0171] This method is directed to the effect of each procyanidin onblood pressure. Rats are instrumented in order to monitor systolic anddiastolic blood pressure. Each of the procyanidins are injectedintravenously (dosage range=100-0.1 μg/kg), and alteration of bloodpressure is assessed. In addition, the effect of each procyanidin on thealteration of blood pressure evoked by epinephrine is determined. Therole of NO is ascertained by the addition of nitroarginine or(1)—N-methyl arginine.

[0172] These studies illustrate that the cocoa procyanidins are usefulin modulating vasodilation, and are further useful with respect tomodulating blood pressure or addressing coronary conditions, andmigraine headache conditions.

Example 8 NO Dependent Hypotension In The Guinea Pig

[0173] The effect of five cocoa procyanidin fractions on guinea pigblood pressure were investigated. Briefly, guinea pigs (approximately400 g body weight; male and female) were anesthetized upon injection of40 mg/kg sodium pentobarbital. The carotid artery was cannulated formonitoring of the arterial blood pressure. Each of the five cocoaprocyanidin fractions was injected intravenously (dose range 0.1mg/kg-100 mg/kg) through the jugular vein. Alterations of blood pressurewere recorded on a polygraph. In these experiments, the role of NO wasascertained by the administration of L-N-methylarginine (1 mg/kg) tenminutes prior to the administration of cocoa procyanidin fractions.

[0174] Cocoa procyanidin fractions were prepared and analyzed accordingto the procedures described in U.S. Pat. No. 5,554,645, herebyincorporated herein by reference.

[0175] Fraction A: Represents a preparative HPLC fraction comprised ofmonomers-tetramers. HPLC analysis revealed the following composition:Monomers 47.2% Dimers 23.7 Trimers 18.7 Tetramers 10.3

[0176] Fraction B: Represents a preparative HPLC fraction comprised ofpentamers-decamers. HPLC analysis revealed the following composition:Pentamers 64.3% Hexamers 21.4 Heptamers 7.4 Octamers 1.9 Nonamers 0.9Decamers 0.2

[0177] Fraction C: Represents an enriched cocoa procyanidin fractionused in the preparation of Fractions A and B (above). HPLC analysisrevealed the following composition: Monomers 34.3% Dimers 17.6 Trimers16.2 Tetramers 12.6 Pentamers 8.5 Hexamers 5.2 Heptamers 3.1 Octamers1.4 Nonamers 0.7 Decamers 0.3

[0178] Fraction D: Represents a procyanidin extract prepared from a milkchocolate. HPLC analysis revealed a composition similar to that listedin the Table 4 for Brand 8. Additionally, caffeine 10% and theobromine6.3% were present.

[0179] Fraction E: Represents a procyanidin extract prepared from a darkchocolate prepared with alkalized liquor. HPLC analysis revealed acomposition similar to that listed in the Table 4 for Brand 12.Additionally, caffeine 16.0% and theobromine 5.8% were present.

[0180] In three separate experiments, the effects of administering 10mg/kg cocoa procyanidin fractions on arterial blood pressure ofanesthetized guinea pigs was investigated. Upon intravenous injection,procyanidin fractions A and E evoked a decrease in blood pressure ofabout 20%. This decrease was only marginally different from thatobtained from a solvent (DMSO) control (15+5%, n=5). In contrast,procyanidin fractions B, C and D (10 mg/kg) induced marked decreases inblood pressure, up to 50-60% for C. In these experiments the order ofhypotensive effect was as follows: C>B>D>>A=E.

[0181] Typical recordings of blood pressure elicited after injection ofprocyanidin fractions appear in FIG. 8A for fraction A and FIG. 8B forfraction C. FIG. 9 illustrates the comparative effects on blood pressureby these fractions.

[0182] The possible contribution of NO in the hypotension in the guineapig induced by administration of fraction C was analyzed usingL-N-methyl arginine (LNMMA). This pharmacological agent inhibits theformation of NO by inhibiting NO synthase. L-NMMA was administered atthe dose of 1 mg/kg, ten minutes prior to injection of the cocoaprocyanidin fractions. As shown in FIG. 10, treatment of the animalswith L-NMMA completely blocked the hypotension evoked by the procyanidinfraction C. Indeed, following treatment with this inhibitor, thealterations of blood pressure produced by fraction C were similar tothose noted with solvent alone.

Example 9 Effect of Cocoa Procyanidin Fractions on NO Production inHuman Umbilical Vein Endothelial Cells

[0183] Human umbilical vein endothelial cells (HUVEC) were obtained fromClonetics and cultures were carried out according to the manufacturer'sspecifications. HUVEC cells were seeded at 5,000 cells/cm² in 12-wellplates (Falcon). After the third passage under the same conditions, theywere allowed to reach confluence. The supernatant was renewed with freshmedium containing defined concentrations of bradykinin (25, 50 and 100nM) or cocoa procyanidin fractions A-E (100 μg/mL) as described inExample 3. The culture was continued for 24 hr. and the cell freesupernatants were collected and stored frozen prior to assessment of NOcontent as described below. In selected experiments, the NO synthase(NOS) antagonist, Nonitro-L-arginine methyl ester (L-NAME, 10 μM) wasadded to assess the involvement of NOS in the observed NO production.

[0184] HUVEC NO production was estimated by measuring nitriteconcentration in the culture supernatant by the Griess reaction. Griessreagent was 1% sulfanilamide, 0.1% N-(1-naphthyl)-ethylenediaminedihydrochloride. Briefly, 50 μL aliquots were removed from the varioussupernatants in quadruplicate and incubated with 150 μL of the Griessreagent. The absorbency at 540 nm was determined in a multiscan(Labsystems Multiskans MCC/340) apparatus. Sodium nitrite was used atdefined concentrations to establish standard curves. The absorbency ofthe medium without cells (blank) was subtracted from the value obtainedwith the cell containing supernatants.

[0185]FIG. 11 illustrates the effect of bradykinin on NO production byHUVEC where a dose dependent release of NO was observed. The inhibitorL-NAME completely inhibited the bradykinin induced NO release.

[0186]FIG. 12 illustrates the effect of the cocoa procyanidin fractionson NO production by HUVEC cells. Fractions B, C and D induced a moderatebut significant amount of NO production by HUVEC. By far, Fraction C wasthe most efficient fraction to induce NO formation as assessed by theproduction of nitrites, while Fraction E was nearly ineffective. Theeffect of Fraction C on NO production was dramatically reduced in thepresence of L-NAME. Interestingly, Fractions B, C and D contained higheramounts of procyanidin oligomers than Fractions A and E. Adistinguishing difference between Fractions D and E was that E wasprepared from a dark chocolate which used alkalized cocoa liquor as partof the chocolate recipe. Alkalization leads to a base catalyzedpolymerization of procyanidins which rapidly depletes the levels ofthese compounds. An analytical comparison of procyanidin levels found inthese types of chocolate appear in the Table 4, where Brand 12 is a darkchocolate prepared with alkalized cocoa liquor and Brand 11 is a typicalmilk chocolate. Thus, extracts obtained from milk chocolates containhigh proportions of procyanidin oligomers which are capable of inducingNO. The addition of the NO inhibitor L-NMMA to the Fraction C sampleclearly led to the inhibition of NO. The results obtained from theprocyanidin fractions were consistent to those observed with thebradykinin induced NO experiment (see FIG. 11).

[0187] As in the case of the HUVEC results, cocoa procyanidin fraction Celicited a major hypotensive effect in guinea pigs, whereas fractions Aand E were the least effective. Again, the presence of high molecularweight procyanidin oligomers were implicated in the modulation of NOproduction.

Example 10 Effect of Cocoa Procyanidin Fractions on Macrophage NOProduction

[0188] Fresh, human heparinized blood (70 mL) was added with an equalvolume of phosphate buffer saline (PBS) at room temperature. AFicoll-Hypaque solution was layered underneath the blood-PBS mixtureusing a 3 mL Ficoll-Hypaque to 10 mL blood-PBS dilution ratio. The tubeswere centrifuged for 30 minutes at 2,000 rpm at 18-20° C. The upperlayer containing plasma and platelets was discarded. The mononuclearcell layer was transferred to another centrifuge tube and the cells werewashed 2×in Hanks Balanced Saline Solution. The mononuclear cells wereresuspended in complete RPMI 1640 supplemented with 10% fetal calfserum, counted, and the viability determined by the Trypan Blueexclusion method. The cell pellet was resuspended in complete RPMI 1640supplemented with 20% fetal calf serum to a final concentration of 1×10⁶cells/mL. Aliquots of the cell suspension were plated into a 96 wellculture plate and rinsed 3× with RPMI 1640 supplemented with 10% fetalcalf serum and the nonadherent cells (lymphocytes) were discarded.

[0189] These cells were incubated for 48 hours in the presence orabsence of five procyanidin fractions described in Example 3. At the endof the incubation period, the culture media were collected, centrifugedand cell free supernatants were stored frozen for nitrate assaydeterminations.

[0190] Macrophage NO production was determined by measuring nitriteconcentrations by the Greiss reaction. Greiss reagent was 1%sulfanilamide, 0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride.Briefly, 50 μL aliquots were removed from the supernatants inquadruplicate and incubated with 150 μL of the Greiss reagent. Theabsorbency at 540 nm was determined in a multiscan (LabsystemsMultiskans MCC/340) apparatus. Sodium nitrite was used at definedconcentrations to establish standard curves. The absorbency of themedium without cells (blank) was subtracted from the value obtained withthe cell containing supernatants.

[0191] In a separate experiment, macrophages were primed for 12 hours inthe presence of 5 U/mL gamma-interferon and then stimulated with 10μg/mL LPS for the next 36 hours in the presence or absence of 100 μg/mLof the five procyanidin fractions.

[0192]FIG. 13 indicates that only procyanidin fraction C, at 100 μg/mL,could induce NO production by monocytes/macrophages. Basal NO productionby these cells was undetectable and no nitrite could be detected in anyof the cocoa procyanidin fractions used at 100 μg/mL. FIG. 14 indicatesthat procyanidin fractions A and D enhanced LPS-induced NO production byy-interferon primed monocytes/macrophages. Procyanidin fraction C wasmarginally effective, since LPS-stimulated monocytes/macrophagescultured in the absence of procyanidin fractions produced only 4μmole/10⁵ cells/48 hours. γ-Interferon alone was ineffective in inducingNO.

[0193] Collectively, these results demonstrate that mixtures of thecocoa procyanidins used at specific concentrations are capable ofinducing monocyte/macrophage NO production both independent anddependent of stimulation by LPS or cytokines.

Example 11 The Effect of Cocoa Procyanidins on Lipoxygenase Activity

[0194] The specific hypothesis which was tested in this part of the workwas that polyphenolic cocoa extracts inhibit the activity oflipoxygenase in a dose-dependent and component specific manner.

[0195] Lipoxygenase Type I-B from soybean, linoleic acid (approx. 99%),Tween 20 (Polyoxyethylene-sorbitan monolaurate), and control phenols:Nordihydroguaiaretic acid (NDGA), (+)-catechin and (−)-epicatechin wereobtained from Sigma Chemical. The cocoa polypenolics functions whichwere tested included defined phenol fractions (monomer to hexamer) andextracts (crude acetone and pentamer enriched).

[0196] Linoleic acid was solubilized using the emulsifier Tween 20according to the method of Grossman & Zakut (Methods Biochem. Anal.25:303-329, 1979). Briefly, linoleic acid was dissolved in absoluteethanol (70 mg/7 mL) and Tween 20 (250 μL) was added. The ethanol wasevaporated from the solution in the dark under vacuum. The residue wasresuspended in 50 mL PBS and the pH adjusted to 8.0 to result in a clearsolution containing linoleic acid (5 mM) and Tween 20 (0.5% vol.).

[0197] Lipoxygenase was dissolved in PBS pH 8.6 at 5000 U/mL. Cocoaphenols were dissolved in pure water and diluted from the followingstock solutions. 1 to 6mer were dissolved at 1 mM, extracts at 1 mg/mL(1 g crude acetone extract/L ˜3.0 mM monomer, 1 g 5mer enrichedextract/L ˜3.4 mM).

[0198] Reaction mixtures contained dissolved linoleic acid (100 μM) inPBS pH 7.4 plus/minus test phenols in water. The reaction was initiatedby adding lipoxygenase for a final concentration of 100 U/mL. Conjugateddiene measurement of hydroperoxide formation of linoleic acid wasmeasured by recording kinetic scans over 5 or 10 minutes on a BeckmanDU-600 UV spectrophotometer (at 37° C., absorbance 234 nm). Inhibitionwas calculated for each sample set of 6 reaction cuvettes (I controlwith water, 5 experimental with phenol in water):

% Inhibition=(Δabs. control−Δabs. experimental)/Δabs. control)×100

[0199] The high UV absorbance of the cocoa components did not allow themeasurement of IC₅₀ at actual concentrations. For this reason an IC₅₀value was calculated by extrapolating the logarithmic regression curve(% inhibition over log phenol concentration) to the concentration atwhich 50% inhibition of lipoxygenase activity was achieved by the testsubstance. Extrapolated IC₅₀ values give a rough approximation of thelipoxygenase inhibitory activity of cocoa polyphenol extracts.

[0200] Results

[0201] NDGA is an established inhibitor of soybean and several mammalianlipoxygenases (Kemal et al, Biochemistry 26:7064-7072, 1987). It iscommercially used as an antioxidant in fats and oils. NDGA serves as apositive control since the IC₅₀ value of 2×10⁻⁴ M was not reached byother test phenols. (+)-catechin and (−) epicatechin are assumed to bestructurally similar to the cocoa polyphenol monomer.

[0202] Phenols were compared on a molar basis. Lipoxygenase inhibitionby (+)-catechin (3×10⁻³M) was two magnitudes stronger than by(−)-epicatechin (3×10⁻³ M) and very similar to inhibition by +cocoamonomer (5×10⁻³M), dimer (2×10⁻³M) and trimer (2×10⁻³M). Lipoxygenaseinhibition by the higher oligomers correlated less well with the log ofthe molar concentration. At final concentrations of 0.3 to 25 μM, thetetramer, pentamer and hexamer inhibited 5-30% lipoxygenase activity.However, phenol concentration and inhibition did not correlatesignificantly. The crude acetone extract exhibited a concentrationdependent lipoxygenase inhibition (IC₅₀=5 μM, compared on a monomerbasis) approximately 3 magnitudes weaker than monomer to trimerfractions. The pentamer enriched extract (IC₅₀=59M) could not be said toinhibit lipoxygenase activity at a meaningful level.

[0203] The soybean lipoxygenase inhibitory activity of cocoa phenolextracts resemble that of (+)-catechin. There is little difference amongthe activities of the monomer to trimer if compared on a molar basis,suggesting a steric inhibition of the enzyme. The inhibition may bespecific for the low molecular weight components (monomer to trimer)since the tetramer to hexamer compounds show considerably lesslipoxygenase inhibitory activity and the pentamer enriched extract isless inhibiting than the crude extract, which contains more of themonomer to trimer oligomeric fractions. Given the above results, wesuggest that cocoa polyphenols may inhibit soybean lipoxygenase eitherby chelating the prosthetic iron ion, or by scavenging free radicals viaphenolic hydroxyl groups in the oxidation reaction, which includes freeradical intermediates. Larger oligomers (the tetramers to hexamers) maybe sterically hindered and thus may not be capable of reading thecatalytic site of the enzyme.

Example 12 The Effect of Cocoa Procyanidins on Cyclo-oxygenase 1 & 2

[0204] The effect of procyanidins on cyclo-oxygenase 1 & 2 (COX-1/COX-2)activities was assessed by incubating the enzymes (derived from ramseminal vesicle and sheep placenta, respectively), with arachidonic acid(5 μM) for 10 minutes at room temperature, in the presence of varyingconcentrations of procyanidin solutions containing monomers to decamersand containing a procyanidin mixture. Turnover was assessed by usingPGE2 EIA kits from Interchim (France). Indomethacin was used as areference compound. The results are presented in the following Table,wherein the IC₅₀ values are expressed in units of μM (except for Sample11, which represents a procyanidin mixture) and where the samples S1 toS110 represent procyanidin oligomers (monomer through decamer), and IC₅₀is expressed in units of mg/mL. SAMPLE RATION IC₅₀ # IC₅₀COX-1*IC₅₀COX-2* COX-2/COX-1 1 0.074 0.197 2.66 2 0.115 0.444 3.86 3 0.2580.763 2.96 4 0.154 3.73 24.22 5 0.787 3.16 4.02 6 1.14 1.99 1.75 7 1.894.06 2.15 8 2.25 7.2 3.20 9 2.58 2.08 0.81 10  3.65 3.16 0.87 11  0.04870.0741 1.52 Indomethacin 0.599 13.5 22.54

[0205]FIGS. 15A and B shows the effects of Indomethacin on COX-1 andCOX-2 activities. FIGS. 16A and B shows the correlation between thedegree of polymerization of the procyanidin and IC₅₀ with COX-1 andCOX-2; FIG. 17 shows the correlation between IC₅₀ values on COX-1 andCOX-2. FIGS. 18A through Y show the IC₅₀ values of each Sample 1-11)with COX—I and COX-2.

[0206] The results indicate that the cocoa procyanidins have analgesic,anticoagulant, and anti-inflammatory utitlities. Since COX-2 has beenlinked to colon cancers, inhibition of COX-2 activity by the cocoaprocyanidins provides a plausible mechanism by which the cocoaprocyanidins have antineoplastic activity against colon cancer.

[0207] COX-1 and COX-2 are also implicated in the synthesis ofprostaglandins. The above show that the cocoa procyanidins can modulaterenal functions, immune responses, fever, pain, mitogenesis, apoptosis,prostaglandin synthesis, ulceration (e.g., gastric), and reproduction.It should be noted that modulation of renal function can affect bloodpressure, again implicating the cocoa procyanidins in modulating bloodpressure, vasodilation, and coronary conditions (e.g., modulation ofangiotensin, bradykinin).

[0208] Reference is made to Seibert et al., PNAS USA 91:12013-12017(December, 1994), Mitchell et al., PNAS USA 90:11693-11697 (December1994), Dewitt et al., Cell 83:345-348 (Nov. 3, 1995), Langenbach et al.,Cell 83:483-92 (Nov. 3, 1995), Sujii et al., Cell 83:493-501 (Nov. 3,1995) and Morham et al., Cell 83:473-82 (Nov. 3, 1995).

[0209] Thus, in addition to having analgesic properties, there may alsobe a synergistic effect by the cocoa procyanidins when administered withother analgesics. Likewise, in addition to having antineoplasticproperties, there may also be a synergistic effect by the cocoaprocyanidins when administered with other antineoplastic agents.

Example 13 The Effects of Cocoa Procyanidin Extracts and CocoaProcyanidin Oligomeric Fractions on Basal Endothelial Cell Release ofProstanoids and Total Endothelin (ET).

[0210] The effect of purified procyanidin monomers and pentamers fromcocoa polyphenol extract on the release of the prostanoids prostacyclin,prostaglandin (PGE2) and thromboxane, and total endothelin on bovine andhuman endothelial cells in culture was studied.

[0211] Altered endothelial cell (EC) release of important signalingmolecules, such as endothelins and prostanoids, could explain somevasoprotective phytochemical effects including beneficial alterations invessel permeability and disposition toward platelet aggregation/thrombusformation.

[0212] Indomethacin was purchased from Cayman (Ann Arbor, Mich. USA).Monomeric and pentameric procyanidin fractions were purified fromprocyanidin-enriched cocoa powder according to the examples described inthis application, and were analysed by the method of Hammerstone et al.,“Identification of Procyanidins in Cocoa, Theobroina cacao, andChocolate Using High-Performance Liquid Chromatography/MassSpectrometry”, J. Agric. Food Chem., 47:2:490-496, 1999. Conformation ofmolecular weight purity was obtained by mass spectrometry.

[0213] Bovine aortic endothelial cells (BAEC) were provided by M. E.O'Donnell of University of California, Davis. Human aortic endothelialcells (HAEC) were purchased from Clonetics (San Diego, Calif.). Thecells were cultured in Eagle's minimum essential medium (EMEM) asdescribed previously by Schramm et al. “Endothelial Cell Basal PGI₂ IsStimulated By Wine In Vitro: One Mechanism That May Mediate TheVasoprotective Effects Of Wine”, J. Nutr. Biochem. 8:647-651, 1997;“Differential Effects Of Small And Large Molecular Weight WinePhytochemicals On Endothelial Cell Eicosanoid Release”, J. Agric. andFood. Chem. 46(5): 1900-1905, 1998; and “Energy Dependent System InMammalian Endothelial Cells For Rapid Flavonoid Up-Take”, J. Nutr.Biochem. In Press, 1999.

[0214] ECs (passage<11) were seeded onto 24-well plates with EMEMcontaining 2 mmol glutamine/L, 10% fetal bovine serum, 100 unitspenicillin/L, 0.1 mg streptomycin/L, and 0.25 μg amphotericin/L.Confluent cells were treated in 250 ml of phenol red-free EMEMcontaining treatment compounds where applicable. Medium incubated withEC's was analyzed after application of procyanidin fractions (10, 20,and 30 pmol/L) and incubation with ECs for 0 and 20 min. The medium wasstored at −70° C. until analyzed by immunoassay as described below. ECintegrity was monitored by Trypan Blue exclusion as described inBioadjieras et al., “Exclusion Of Trypan Blue From Microcarriers ByEndothelial Cells: An In Vitro Barrier Function Test”, Methods in Lab.Invest. 50:239-246, 1984.

[0215] Immunoassay procedures were conducted as described by Westcott etal, “Analysis Of 6-Keto PGF1 Alpha, 5-HETE, And LTC4 In Rat Lung:Comparison Of GM/MS, RIA and EIA”, Prostaglandins 32:857-873, 1986;Yakota et al, “Enzyme Immunoassay Of Prostanoids In Blood And Urine”,Adv. Prostgl. Thrombox. Leukot. Res. 15:33-34, 1985; Schramm et al,1997; 1998;1999. Medium total ET (ET-I+ET-2+ET-3) was measured withCayman immunoassay #583151. The prostacyclin (PGI₂) metabolite 6-ketoprostaglandin F1-alpha was measured with Cayman enzyme immunoassay#515211, the thromboxane (TXA₂) metabolite TXB₂ was measured with Caymanenzyme immunoassay 519031, and PGE₂ was determined with Caymanimmunoassay #514016.

[0216] An endothelial cell (EC) monolayer culture system was used tocompare the effects of the purified cocoa procyanidin monomers andpentamers in a system that closely mimics the EC monolayer of a bloodvessel. The established assay conditions mimic those used previously toshow that a wine fraction with mass <3000 da. induced different effectson EC prostanoid synthesis than did a wine fraction having a massof >3000 (Schramm et al, “Differential Effects Of Small And LargeMolecular Weight Wine Phytochemicals On Endothelial Cell EicosanoidRelease”, J. Agric. and Food. Chem. 46(5): 1900-1905, (1998). Theeffects on basal cell function were examined. Neither cell viability norcell morphology was effected by cocoa procyanidin treatments. Eachmilliliter of control medium incubated with BAECs for 20 min contained73.5=+/0.044 ng TXB2 and 294+/−6.3 pg PGE2.

[0217] As shown in FIG. 19, the addition of the monomeric or pentamericcocoa procyanidins to medium incubated with BAECs for 20 min alteredmedium prostanoid and ET concentrations when compared to the controlmedium alone. Media containing either monomeric (A¹) or pentameric (A²)procyanidins contained more 6-keto-PGF¹-alpha than the control medium.Medium PGE² was reduced by the monomeric fraction (B1) in adose-dependent manner and by the pentameric fraction (B12) at 30 (μM. Nosignificant effect of either cocoa procyanidin fraction was noted onmedium TXB (C₁₋₂). Although the monomeric and pentameric cocoaprocyanidin fractions had similar effects on medium 6-keto-PGF¹-alphaand PGE², they had opposite effects on medium ET, with the monomericfraction (D¹) increasing the concentration of ET in the culture mediumat 30 μM and the pentameric fraction (D²) decreasing medium ETconcentration in a dose dependent manner.

[0218] Data in FIGS. 20A and 20B demonstrate the similar manner in whichmonomeric and pentameric cocoa procyanidins affected aortic ECs fromcows (BAEC) and humans (HAEC).

[0219] The above data demonstrate that the procyanidins present in cocoacan induce eicosanoid and endothelin effects which promote a state ofvessel relaxation and decreased platelet aggregation/thrombus formation,i.e., induction of prostacyclin and inhibition of endothelin andprostaglandin. The cocoa procyanidin extracts, either as dietarycomponents or in acceptable pharmacological form, should be useful invasoprotective prophylaxis and treatment for vascular disease.

Example 14 The Effects of the Consumption of a Procyanidin-EnrichedCocoa Beverage on Platelet Activity

[0220] The effects of consumption of a cocoa beverage on modulation ofplatelet activation and primary haemostasis were studied.

[0221] Thirty healthy, non-smoking adults with no history of heartdisease or haemostatic disorders participated in the study. Venous bloodwas obtained from 10 subjects (4 males and 6 females, 24-49 years ofage) who consumed a cocoa beverage, 10 subjects (4 males and 5 females,26-50 years of age) who consumed a caffeine beverage as a control, and10 subjects (4 males, 6 females, 24-50 years of age) who consumed wateras a control. All women were premenopausal and were not takingestrogens. Participants were instructed to abstain from nonsteroidal,anti-inflammatory medication for at least 4 days, from alcoholicbeverages for at least 2 days, and from caffeine- ortheobromine-containing foods for at least 24 hours before the test andduring the test day.

[0222] Blood was obtained from each test and control subject between 8and 10 μM in two 5-ml evacuated tubes containing 0.5 ml of 3.2% bufferedsodium citrate solution (Becton Dickinson, Franklin Lakes, N.J.).Specimens obtained as the result of a traumatic venipuncture and/orthose with obvious clots were not analyzed. Test subjects then drank 300ml of a beverage containing 18.75 g of procyanidin enriched cocoa powderand 12.5 g of sucrose mixed with distilled water (see Adamson, G. E.,Lazarus, S. A., Mitchell, A. E., Prior R. L., Cao, G., Jacobs, P. H.,Kremers B. G., Hammerstone, J. F., Rucker R., Ritter K. A., Schmitz H.H., HPLC Method for the Quantification of Procyanidins in Cocoa andChocolate Samples and Correlation to Total Antioxidant Capacity, J Ag.Food Chem.; 1999; 47 (10) 4184-4188). The cocoa powder providedapproximately 960 mg of total procyanidins, 17 mg caffeine and 285 mgtheobromine (see Clapperton, J., Hammerstone, J. F., Romanczyk, L. J.,Yow, S., Lim, D., Lockwood, R., Polyphenols and Cocoa Flavour,Proceedings, 16th International Conference of Groupe Polyphenols,Lisbon, Portugal, Groupe Polyphenols: Norbonne, France, 1992; Tome II,pp. 112-115.). Control subjects drank either a beverage containing 17 mgcaffeine and 12.5 g sucrose or plain water. Additional blood sampleswere obtained 2 and 6 hours after consumption of the beverages. Onefemale subject was not present for the 6-hour blood draw after cocoaconsumption.

[0223] Procyanidins were quantified as follows: a composite standard wasmade using commercially available (−)-epicatechin for the monomer.Dimers through decamers were obtained in a purified state by the methodsdescribed in Hammerstone, J. F. et al., “Identification of Procyanidinsin Cocoa (Theobroma cacao) and Chocolate Using High-Performance LiquidChromatography/Mass Spectrometry”, J. Ag. Food Chem.; 1999; 47 (10)490-496, Lazarus, S. A. et al., High-performance LiquidChromatography/Mass Spectrometry Analysis of Proanthocyanidins in Foodsand Beverages, J. Ag. Food Chem.; 1999; 47 (9); 3693-3701 and Adamson,G. E. et al., “HPLC Method for the Quantification of Procyanidins inCocoa and Chocolate Samples and Correlation to Total AntioxidantCapacity”, J. Ag. Food Chem.; 1999; 47 (10) 4184-4188. Standard Stocksolutions using these compounds were analyzed using the normal-phaseHPLC method described previously with fluorescence detection atexcitation and emission wavelengths of 276 nm and 316 nm, respectively.Peaks were grouped and their areas summed to include contributions fromall isomers within any one class of oligomers and calibration curvesgenerated using a quadratic fit. Monomers and smaller oligomers hadalmost linear plots which is consistent with prior usage of linearregression to generate monomer-based and dimer-based calibration curves.

[0224] These calibration curves were then used to calculate procyanidinlevels in samples prepared as follows: First, the cocoa or chocolatesample (about 8 grams) was defatted using three hexane extractions (45mL each). Next, one gram of defatted material was extracted with 5 mL ofthe acetone/water/acetic acid mixture (70:29.5:0.5 v/v). The quantity ofprocyanidins in the defatted material was then determined by comparingthe HPLC data from the samples with the calibration curves obtained asdescribed above (which used the purified oligomers). The percentage offat for the samples (using a one gram sample size for chocolate orone-half gram sample size for liquors) was determined using astandardized method by the Association of Official Analytical Chemists(AOAC Official Method 920.177). The quantity of total procyanidin levelsin the original sample (with fat) was then calculated. Calibration wasperformed prior to each sample run to protect against column-to-columnvariations.

[0225] Within 10 minutes of draw, whole blood was incubated inpolystyrene tubes for 5 minutes at room temperature with 10 μl HEPESbuffer (pH 7.4, unstimulated control), 20 or 100 μM ADP or 20 μMepinephrine (BioData, Horsham, Pa.) in the presence or absence of thepeptide Arg-Gly-Asp-Ser (Sigma, St. Louis, Mo.). After 5 minutes,samples were suspended in 1 ml HEPES buffer and 100 μl of sample weretransferred to tubes containing saturating concentrations (20 μL) eachof the following fluorescent-labeled monoclonal antibodies:PAC1-fluorescein isothiocyanate (FITC), anti-CD62P-phycoerythrin (PE)and anti-CD42a-PerCP. PAC1 recognizes the activated conformation of thefibrinogen-binding receptor GPIIb-IIIa and anti-CD62P recognizesP-selectin, present on the surface of activated platelets. Anti-CD42arecognizes GPIb-IX, which is on the surface of both activated andresting platelets. Mouse IgG, FITC and mouse IgG, PE were used asisotype controls. The Arg-Gly-Asp-Ser-peptide was used to block bindingof the PACI antibody to platelets and thus set the negative controlmarker on the flow cytometer. Antibodies and isotype controls werepurchased from Becton Dickinson Immunocytometry Systems, Inc., San Jose,Calif.

[0226] Whole blood samples in the presence and absence of the agonistsADP and epinephrine were incubated with monoclonal antibodies or isotypecontrol for 20 minutes in the dark at room temperature. Samples werethen fixed in filtered 1% paraformaldehyde (pH 7.2) and stored in thedark at 2-8° C. All samples were analyzed within 48 hours on a FACScanflow cytometer using LYSYS II software. The flow cytometer performancewas verified using 1, 2 and 10 Tm calibration beads (Becton DickinsonImmunocytometry Systems, Inc., San Jose, Calif. and Flow CytometrySystems, Research Triangle Park, N.C.). Twenty-thousand events werecollected in list mode with all light-scatter and fluorescenceparameters in logarithmic mode. Platelets were gated on the basis oflightscatter and CD42a expression. Activated platelets were defined asthe percentage of CD42a positive events coexpressing the activatedconformation of GPIIb-IIIa or P-selectin. Platelet microparticles weredefined as the percentage of CD42a positive events less than 2 μm insize.

[0227] One blood sample drawn at each of the three study time points wasanalyzed within four hours using a platelet function analyzer (PFA-100™,Dade Behring International, Miami, Fla.) according to the manufacturer'sdirections. The PFA-100™ is designed to measure collagen-ADP-andcollagen-epinephrine-stimulated platelet function under shear conditionssimulating those that exist in a small blood vessel (Mammen et al,“PFA-100 System: A New Method For Assessment Of Platelet Disfunction”.Sem. Thromb. Hemostas. 24:195-202, 1998; Fressinaud et al, “Screeningfor Von Willebrand Disease with a New Analyzer using High Shear Stress:a Study of 60 Cases”, Blood 91:1325-31, 1998). Function was measured asa closure time in seconds, which is defined as the time required forblood to occlude an aperture in the test cartridge membrane.

[0228] Data from each treatment or control group were analyzed fordifferences using Friedman's repeated measures ANOVA on ranks (SigmaStatfor Windows, SPSS, Richmond, Calif.). Student-Newman-Keuls multiplecomparison method was used to identify differences between baseline and2 and 6 hours post-consumption results. P values less than 0.05 wereconsidered statistically significant.

[0229] Cocoa consumption suppressed unstimulated (P=0.035, FIG. 21A) andex vivo epinephrine-induced (P=0.008, FIG. 21B) activated GPIIb-IIIaexpression at 2 and 6 hours after ingestion. The median percentages ofplatelets expressing activated GPIIb-IIIa without stimulation were 0.9,0.5 and 0.3% and in response to epinephrine were 9.6, 6.8 and 3.3% attimes zero, 2 and 6 hours, respectively, post consumption. In contrast,there was an increase in epinephrine-stimulated activated GPIIb-IIIaexpression in the control group that drank the caffeine beverage(P=0.048, median=5.3, 6.5 and 7.5% at times zero, 2 and 6 hours postconsumption). There was no change in the control group that drank water.

[0230] The cocoa decreased 20 μM ADP-induced activated GPIlb-IIIaexpression on platelets 2 and 6 hours after consumption (P<0.001, FIG.21C, median=58.5, 44.2 and 38.8% at times zero, 2 and 6 hours postconsumption, respectively). The trend suggested decreased activatedGPIIb-IIIa expression on platelets after cocoa consumption whenactivation was induced by 100 pm ADP (P=0.067, median=76.5, 68.7 and57.6% at times zero, 2 and 6 hours post consumption, respectively).There was no change in ADP-induced activated GPIIb-IIIa expression ingroups consuming the caffeine beverage and water controls.

[0231] A non-significant trend toward decreased P-selectin expressionwas observed after cocoa consumption (P=0.053, FIG. 22A). Cocoaconsumption decreased 20 μM ADP-induced P-selectin expression 2 and 6hours after consumption (P=0.007, FIG. 22C) and 100 μM ADP-inducedP-selectin were 56.1, 54.7 and 41.7% at time zero, 2 and 6 hours postconsumption, respectively.

[0232] There was no evidence of platelet stimulation or inhibition inthe control groups that consumed the caffeine-containing beverage orwater.

[0233] The number of platelet microparticles detected by flow cytometryafter consumption of the cocoa beverage was decreased from baseline at 2hours and was further decreased at 6 hours (see Table 6). In contrast,the number of platelet microparticles was increased at 2 and 6 hoursafter consumption of water and at 6 hours after consumption of thecaffeine-containing beverage. Platelet microparticles arehaemostatically active, phospholipid rich microvesicles that are formedduring physiologic platelet activation.

[0234] Six hours following consumption of the cocoa beverage,collagen-epinephrine-induced closure time was prolonged (see Table 7).This indicates delayed platelet-related primary haemostasis with cocoaconsumption. A trend toward prolonged closure time was observed aftercollagen-ADP-induction (p=0.097); closure time was not changed in thecaffeine control group.

[0235] The results showed that consumption of the chocolate beveragemodified platelet function in humans. First, platelet activationmeasured by platelet activation marker expression in response to weakagonists in vitro was decreased following cocoa consumption. Second,platelet microparticle formation was decreased following cocoaconsumption. And third, the cocoa consumption caused an aspirin-likeeffect on platelet function as measured by platelet-related primaryhaemostasis. The fact that the caffeine beverage control caused anincrease in epinephrine-induced activated GPIIb-IIIa expression andmicroparticle formation would imply that the cocoa procyanidins presentin the cocoa beverage are responsible for the inhibition of plateletactivation and function. TABLE 6 MICROPARTICLE FORMATION AFTERCONSUMPTION OF A COCOA BEVERAGE.⁼ TIME WATER CAFFEINE BEVERAGE COCOABEVERAGE BEFORE CONSUMPTION 0.7 (0.3-1.2) 1.0 (0.6-1.8) 1.9 (1.0-5.0) 2H POST CONSUMPTION 1.2 (0.7-1.6)* 1.1 (0.6-2.0) 1.0 (0.7-1.4)* 6 H POSTCONSUMPTION 1.3 (0.8-2.1)* 1.5 (1.2-2.3)* 0.6 (0.4-1.1)*

[0236] TABLE 7 PLATELET FUNCTION ANALYSIS.= COLLAGEN- EPINEPHRINECOLLAGEN-ADP COCOA CAFFEINE COCOA CAFFEINE TIME BEVERAGE BEVERAGEBEVERAGE BEVERAGE BEFORE CONSUMPTION 125 (61-80) 104 (61-180) 83(60-133) 77 (52-95) 2 H POST CONSUMPTION 135 (82-194) 113 (81-141) 96(65-132) 78 (58-99) 6 H POST CONSUMPTION 164 (101-262)* 114 (79-143) 94(66-116) 82 (67-114)

Example 15 Tablet Formulations

[0237] A tablet formulation was prepared using high cocoa procyanidincocoa solids obtained by methods described above, hereby incorporatedherein by reference. Briefly, this edible material is prepared by aprocess which enhances the natural occurrence of the cocoa procyanidinsin contrast to their levels found in traditionally processed cocoa, suchthat the ratio of the initial amount of the cocoa procyanidins found inthe unprocessed bean to that obtained after processing is less than orequal to 2. For simplicity, this cocoa solids material is designatedherein as CP-cocoa solids. The inventive compound or compounds, e.g., inisolated and/or purified form may be used in tablets as described inthis Example, instead of or in combination with CP-cocoa solids.

[0238] A tablet formula comprises the following (percentages expressedas weight percent): CP-cocoa solids 24.0% 4-Fold Natural vanilla extract(Bush Boake Allen) 1.5% Magnesium stearate (dry lubricant) 0.5%(AerChem, Inc.) Dipac tabletting sugar (Amstar Sugar Corp.) 37.0%Xylitol (American Xyrofin, Inc.) 37.0% 100.0%

[0239] The CP-cocoa solids and vanilla extract are blended together in afood processor for 2 minutes. The sugars and magnesium stearate aregently mixed together, followed by blending in the CP-cocoasolids/vanilla mix. This material is run through a Manesty Tablet Press(B3B) at maximum pressure and compaction to produce round tablets (15mm×5 mm) weighing 1.5-1.8 gram. Another tablet of the above mentionedformula was prepared with a commercially available low fat natural cocoapowder (11% fat) instead of the CP-cocoa solids (11% fat). Both tabletformulas produced products having acceptable flavor characteristics andtexture attributes.

[0240] An analysis of the two tablet formulas was performed using theprocedures described in Example 4. In this case, the analysis focused onthe concentration of the pentamer and the total level of monomers andcocoa procyanidins where n is 2 to 12 which are reported below. pentamertotal pentamer (Tg/ total (Tg/ Tablet sample (Tg/g) (Tg/g) 1.8 gserving) 1.8 g serving) tablet with CP- 239 8,277 430 14,989 cocoasolids tablet with ND 868 ND 1563 commercial low fat cocoa powder

[0241] The data clearly showed a higher level of pentamer and totallevel of cocoa procyanidins in the CP-cocoa solids tablet than in theother tablet formula. Thus, tablet formulas prepared with CP-cocoasolids are an ideal delivery vehicle for the oral administration ofcocoa procyanidins, for pharmaceutical, supplement and foodapplications.

[0242] The skilled artisan in this area can readily prepare other tabletformulas covering a wide range of flavors, colors, excipients, vitamins,minerals, OTC medicaments, sugar fillers, UV protectants (e.g., titaniumdioxide, colorants, etc.), binders, hydrogels, and the like except forpolyvinyl pyrrolidone which would irreversibly bind the cocoaprocyanidins or combination of compounds. The amount of sugar fillersmay be adjusted to manipulate the dosages of the cocoa procyanidins orcombination of compounds.

[0243] Many apparent variations of the above are self-evident andpossible without departing from the spirit and scope of the example.

Example 16 Capsule Formulations

[0244] A variation of Example 15 for the oral delivery of the cocoaprocyanidins is made with push-fit capsules made of gelatin, as well assoft sealed capsules made of gelatin and a plasticizer such as glycerol.The push-fit capsules contain the compound of the invention orcombination of compounds or CP-cocoa solids as described in Example 15in the form of a powder which can be optionally mixed with fillers suchas lactose or sucrose to manipulate the dosages of the cocoaprocyanidins. In soft capsules, the compound of the invention orcombination of compounds or CP-cocoa solids are suspended in a suitableliquid such as fatty oils or cocoa butter or combinations therein. Sincean inventive compound or compounds may be light-sensitive, e.g.,sensitive to UV, a capsule can contain UV protectants such as titaniumdioxide or suitable colors to protect against UV. The capsules can alsocontain fillers such as those mentioned in the previous Example.

[0245] Many apparent variations of the above are self-evident andpossible to one skilled in the art without departing from the spirit andscope of the example.

Example 17 Standard of Identity (SOI) and Non-Standard of Identity(non-SOI) Dark and Milk Chocolate Formulations

[0246] Formulations of the cocoa procyanidins or combination ofcompounds derived by methods embodied in the invention can be preparedinto SOI and non-SOI dark and milk chocolates as a delivery vehicle forhuman and veterinary applications. Reference is made to copending U.S.application Ser. No. 08/709,406, filed Sep. 6, 1996, hereby incorporatedherein by reference. U.S. Ser. No. 08/709,406 relates to a method ofproducing cocoa butter and/or cocoa solids having conserved levels ofthe cocoa procyanidins from cocoa beans using a unique combination ofprocessing steps. Briefly, the edible cocoa solids obtained by thisprocess conserves the natural occurrence of the cocoa procyanidins incontrast to their levels found in traditionally processed cocoa, suchthat the ratio of the initial amount of the cocoa procyanidins found inthe unprocessed bean to that obtained after processing is less than orequal to 2. For simplicity, this cocoa solids material is designatedherein as CP-cocoa solids. The CP-cocoa solids are used as a powder orliquor to prepare SOI and non-SOI chocolates, beverages, snacks, bakedgoods, and as an ingredient for culinary applications.

[0247] The term “SOI chocolate” as used herein shall mean any chocolateused in food in the United States that is subject to a Standard ofIdentity established by the U.S. Food and Drug Administration under theFederal Food, Drug and Cosmetic Act. The U.S. definitions and standardsfor various types of chocolate are well established. The term “non-SOIchocolate” as used herein shall mean any nonstandardized chocolateswhich have compositions which fall outside the specified ranges of thestandardized chocolates.

[0248] Examples of nonstandardized chocolates result when the cocoabutter or milk fat are replaced partially or completely; or when thenutrative carbohydrate sweetener is replaced partially or completely; orflavors imitating milk, butter, cocoa powder, or chocolate are added orother additions or deletions in the formula are made outside the U.S.FDA Standards of Identity for chocolate or combinations thereof.

[0249] As a confection, chocolate can take the form of solid pieces ofchocolate, such as bars or novelty shapes, and can also be incorporatedas a component of other, more complex confections where chocolate isoptionally combined with any Flavor & Extract Manufacturers Association(FEMA) material, natural juices, spices, herbs and extracts categorizedas natural-flavoring substances; nature-identical substances; andartificial flavoring substances as defined by FEMA GRAS lists, FEMA andFDA lists, Council of Europe (CoE) lists, International Organization ofthe Flavor Industry (IOFI) adopted by the FAO/WHO Food StandardProgramme, Codex Alimentarius, and Food Chemicals Codex and generallycoats other foods such as caramel, nougat, fruit pieces, nuts, wafers orthe like. These foods are characterized as microbiologicallyshelf-stable at 65-85[ ]F under normal atmospheric conditions. Othercomplex confections result from surrounding with chocolate softinclusions such as cordial cherries or peanut butter. Other complexconfections result from coating ice cream or other frozen orrefrigerated desserts with chocolate. Generally, chocolate used to coator surround foods must be more fluid than chocolates used for plainchocolate solid bars or novelty shapes.

[0250] Additionally, chocolate can also be a low fat chocolatecomprising a fat and nonfat solids, having nutrative carbohydratesweetener(s), and an edible emulsifier. As to low fat chocolate,reference is made to U.S. Pat. Nos. 4,810,516, 4,701,337, 5,464,649,5,474,795, and WO 96/19923.

[0251] Dark chocolates derive their dark color from the amount ofchocolate liquor, or alkalized liquor or cocoa solids or alkalized cocoasolids used in any given formulation. However, the use of alkalizedcocoa solids or liquor would not be used in the dark chocolateformulations in the invention.

[0252] Examples of formulations of SOI and non-SOI dark and milkchocolates are listed in Tables 8 and 9. In these formulations, theamount of the cocoa procyanidins present in CP-cocoa solids was comparedto the cocoa procyanidins present in commercially available cocoasolids.

[0253] The following describes the processing steps used in preparingthese chocolate formulations.

[0254] Process for Non-SOI Dark Chocolate

[0255] 1. Keep all mixers and refiners covered throughout process toavoid light.

[0256] 2. Batch all the ingredients excluding 40% of the free fat (cocoabutter and anhy. milk fat) maintaining temperature between 30-35° C.

[0257] 3. Refine to 20 microns.

[0258] 4. Dry conche for 1 hour at 35° C.

[0259] 5. Add full lecithin and 10% cocoa butter at the beginning of thewet conche cycle; wet conche for 1 hour.

[0260] 6. Add all remaining fat, standardize if necessary and mix for 1hour at 35° C.

[0261] 7. Temper, mould and package chocolate.

[0262] Process for SOI Dark Chocolate

[0263] 1. Batch all ingredients excluding milk fat at a temperature of60° C.

[0264] 2. Refine to 20 microns.

[0265] 3. Dry conche for 3.5 hours at 60° C.

[0266] 4. Add lecithin and milk fat and wet conche for 1 hour at 60° C.

[0267] 5. Standardize if necessary and mix for 1 hour at 35° C.

[0268] 6. Temper, mould and package chocolate.

[0269] Process for non-SOI Milk Chocolate

[0270] 1. Keep all mixers and refiners covered throughout process toavoid light.

[0271] 2. Batch sugar, whole milk powder, malted milk powder, and 66% ofthe cocoa butter, conche for 2 hours at 75° C.

[0272] 3. Cool batch to 35° C. and add cocoa powder, ethyl vanillin,chocolate liquor and 21% of cocoa butter, mix 20 minutes at 35° C.

[0273] 4. Refine to 20 microns.

[0274] 5. Add remainder of cocoa butter, dry conche for 1.5 hour at 35°C.

[0275] 6. Add anhy. milk fat and lecithin, wet conche for 1 hour at 35°C.

[0276] 7. Standardize, temper, mould and package the chocolate.

[0277] Process for SOI Milk Chocolate

[0278] 1. Batch all ingredients excluding 65% of cocoa butter and milkfat at a temperature of 60° C.

[0279] 2. Refine to 20 microns.

[0280] 3. Dry conche for 3.5 hours at 60° C.

[0281] 4. Add lecithin, 10% of cocoa butter and anhy. milk fat; wetconche for 1 hour at 60° C.

[0282] 5. Add remaining cocoa butter, standardize if necessary and mixfor 1 hour at 35° C.

[0283] 6. Temper, mould and package the chocolate.

[0284] The CP-cocoa solids and commercial chocolate liquors used in theformulations were analyzed for the pentamer and total level of monomersand cocoa procyanidins where n is 2 to 12 prior to incorporation in theformulations. These values were then used to calculate the expectedlevels in each chocolate formula as shown in Tables 8 and 9. In thecases for the non-SOI dark chocolate and non-SOI milk chocolate, theirproducts were similarly analyzed for the pentamer, and the total levelof monomers and the cocoa procyanidins where n is 2 to 12. The resultsappear in Tables 8 and 9.

[0285] The results from these formulation examples indicated that SOIand non-SOI dark and milk chocolates formulated with CP-cocoa solidscontained approximately 6.5 times more expected pentamer, and 3.5 timesmore expected total levels in the SOI and non-SOI dark chocolates; andapproximately 4.5; 7.0 times more expected pentamer and 2.5; 3.5 timesmore expected total levels in the SOI and non-SOI milk chocolates,respectively.

[0286] Analyses of some of the chocolate products were not performedsince the difference between the expected levels of the cocoaprocyanidins present in finished chocolates prepared with CP-cocoasolids were dramatically higher than those formulas prepared withcommercially available cocoa solids. However, the effects of processingwas evaluated in the non-SOI dark and milk chocolate products. As shownin the tables, a 25-50% loss of the pentamer occurred, while slightdifferences in total levels were observed. Without wishing to be boundby any theory, it is believed that these losses are due to heat and/orlow chain fatty acids from the milk ingredient (e.g. acetic acid,propionic acid and butyric acid) which can hydrolyze the oligomers (e.g.a trimer can hydrolyze to a monomer and dimer). Alternatively, timeconsuming processing steps can allow for oxidation or irreversiblebinding of the cocoa procyanidins to protein sources within the formula.Thus, the invention comprehends altering methods of chocolateformulation and processing to address these effects to prevent orminimize these losses.

[0287] The skilled artisan will recognize many variations in theseexamples to cover a wide range of formulas, ingredients, processing, andmixtures to rationally adjust the naturally occurring levels of thecocoa procyanidins for a variety of chocolate applications. TABLE 8 DarkChocolate Formulas Prepared with non-Alkalized Cocoa Ingredients Non-SOIDark SOI Dark SOI Dark Chocolate Chocolate Chocolate Using UsingCommercial Using CP-cocoa solids CP-Cocoa Solids Cocoa SolidsFormulation: Formulation: Formulation 41.49% Sugar 41.49% sugar 41.49%sugar 3% whole milk 3% whole milk 3% whole milk powder powder powder 26%CP-cocoa solids 52.65% CP-liquor 52.65% com. liquor 4.5% com. liquor2.35% anhy. milk fat 2.35% anhy. milk fat 21.75% cocoa butter 0.01%vanillin 0.01% vanillin 2.75% anhy. milk fat 0.5% lecithin 0.5% lecithin0.01% vanillin 0.5% lecithin Total fat: 31% Total fat: 31% Total fat:31% Particle size: 20 Particle size: 20 Particle size: 20 micronsmicrons microns

[0288] Expected Levels of pentamer and total oligomeric procyanidins(monomers and n=2-12; units of μg/g) Pentamer: 1205 Pentamer: 1300Particle size: 20 microns Total: 13748 Total: 14646 Total: 3948

[0289] Actual Levels of pentamer and total oligomeric procyanidins(monomers and n=2-12; units of μg/g) Pentamer: 561 Not performed Notperformed Total: 14097

[0290] TABLE 9 Milk Chocolate Formulas Prepared with non-Alkalized CocoaIngredients Non-SOI Dark SOI Dark SOI Milk Chocolate Chocolate ChocolateUsing Using Commercial Using CP-cocoa solids CP-Cocoa Solids CocoaSolids Formulation: Formulation: Formulation 46.9965% Sugar 46.9965%Sugar 46.9965% Sugar 15.5% whole 15.5% whole milk 15.5% whole milk milkpowder powder powder 4.5% CP-cocoa solids powder 13.9% com. liquor 5.5%com.liquor 13.9% CP-liquor 1.60% anhy. milk fat 21.4% cocoa butter 1.6%anhy. milk fat 0.0035% vanillin 1.6% anhy. milk fat 0.0035% vanillin0.5% Lecithin 0.035% vanillin 0.5% lecithin 17.5% cocoa butter 0.5%lecithin 17.5% cocoa butter 4.0% malted milk 4.0% malted milk 4.0%malted powder powder powder Total fat: 31.75% Total fat: 31.75% Totalfat: 31.75% Particle size: 20 Particle size: 20 Particle size: 20microns microns microns

[0291] Expected Levels of pentamer and total oligomeric procyanidins(monomers and n=2-12; units of μg/g) Pentamer: 225 Pentamer: 343Particle size: 49 Total: 2734 Total: 3867 Total: 1042

[0292] Actual Levels of pentamer and total oligomeric procyanidins(monomers and n=2-12; units of μg/g) Pentamer: 163 Not performed Notperformed Total: 2399

Example 18 The Effects of the Oral Consumption of Cocoa on theInhibition of Vascular Endothelium Dependent Relaxation (EDR) byCholesterol

[0293] It has been shown that some plant extracts containing flavonoidsinduce EDR in vitro in rabbit aortas. We studied the effects of chronicoral administration of cocoa on EDR and its protective activity againstthe loss of EDR that occurs with cholesterol feeding. New Zealand Whiterabbits were fed 4 diets for 7 weeks: (1) chow, (2) chow+200 mgcocoa/day, (3) 2% cholesterol for 3 weeks followed by 4 weeks of chow,(4) 2% cholesterol for 3 weeks followed by 4 weeks of chow+200 mgcocoa/day. EDR was measured on aortic rings suspended in organ baths (20ml). The rings were pre-contracted with norepinephrine (NE) (10→5M). EDRto acetylcholine (Ach) and cocoa pentamer extract (10→7-10⁻⁵M) wasmeasured as % relaxation to NE. Serum Cholesterol EDR to Pentamer Diet(3 wks) EDR to Ach (%) extract (%) 1(n = 6)  52 ± mg/dL 49.0 ± 5.1 46.5± 4.5 2(n = 5)  30 ± 4 mg/dL 44.4 ± 5.1 44.5 ± 3.6 3(n = 9) 1377 ± 218mg/dL 16.1 ± 5.0(n = 6)* 25.5 ± 9.7(n = 3) 4(n = 5) 1084 ± 266 mg/dL42.4 ± 11.3 49.5 ± 5.7

[0294] These results would indicate that oral administration of cocoapowder protects against the loss of EDR in cholesterol fed rabbits.

1-33. (Cancelled)
 34. A method of anti-platelet therapy or prophylaxiscomprising administering to a subject in need thereof an effectiveamount of a procyanidin oligomer comprising from 2 to 18 monomeric unitsof the following formula:

or a derivative thereof; wherein the monomeric units are connected viainterflavan linkages 4→6 and/or 4→8, and the subject is a human or aveterinary animal.
 35. The method of claim 34, wherein the derivative isa gallated procyanidin oligomer.
 36. The method of claim 34, wherein theprocyanidin oligomer comprises from 3 to 12 monomeric units.
 37. Themethod of claim 34, wherein the procyanidin oligomer comprises from 2 to5 monomeric units.
 38. The method of claim 34, wherein the procyanidinoligomer comprises 2 monomeric units.
 39. The method of claim 34,wherein the procyanidin oligomer has the formula:

and n is 0 to
 16. 40. The method of claim 34, wherein the procyanidinoligomer has the formula:

wherein A and B are independently oligomers having 1 to 15 monomericunits, and the total number of monomeric units in the procyanidinoligomer is 3 to
 18. 41. The method of claim 34, wherein the subject isa human.
 42. The method of claim 35, wherein the subject is a human. 43.The method of claim 36, wherein the subject is a human.
 44. The methodof claim 37, wherein the subject is a human.
 45. The method of claim 38,wherein the subject is a human.
 46. The method of claim 39, wherein thesubject is a human.
 47. The method of claim 40, wherein the subject is ahuman.
 48. The method of claim 41, wherein the human suffers fromatherosclerosis.
 49. The method of claim 42, wherein the human suffersfrom atherosclerosis.
 50. The method of claim 43, wherein the humansuffers from atherosclerosis.
 51. The method of claim 44, wherein thehuman suffers from atherosclerosis.
 52. The method of claim 45, whereinthe human suffers from atherosclerosis.
 53. The method of claim 46,wherein the human suffers from atherosclerosis.
 54. The method of claim47, wherein the human suffers from atherosclerosis.
 55. The method ofclaim 41, wherein the human is at risk of atherosclerosis.
 56. Themethod of claim 42, wherein the human is at risk of atherosclerosis. 57.The method of claim 43, wherein the human is at risk of atherosclerosis.58. The method of claim 44, wherein the human is at risk ofatherosclerosis.
 59. The method of claim 45, wherein the human is atrisk of atherosclerosis.
 60. The method of claim 46, wherein the humanis at risk of atherosclerosis.
 61. The method of claim 47, wherein thehuman is at risk of atherosclerosis.
 62. The method of claim 34, whereinthe procyanidin oligomer is administered with a pharmaceuticallyacceptable carrier.
 63. The method of claim 34, wherein the procyanidinoligomer is in a food composition.
 64. The method of claim 34, whereinthe procyanidin oligomer is in a dietary supplement composition.
 65. Themethod of claim 36, wherein the procyanidin oligomer is administeredwith a pharmaceutically acceptable carrier.
 66. The method of claim 36,wherein the procyanidin oligomer is in a food composition.
 67. Themethod of claim 36, wherein the procyanidin oligomer is in a dietarysupplement composition.
 68. The method of claim 37, wherein theprocyanidin oligomer is administered with a pharmaceutically acceptablecarrier.
 69. The method of claim 37, wherein the procyanidin oligomer isin a food composition.
 70. The method of claim 37, wherein theprocyanidin oligomer is in a dietary supplement composition.
 71. Themethod of claim 38, wherein the procyanidin oligomer is administeredwith a pharmaceutically acceptable carrier.
 72. The method of claim 38,wherein the procyanidin oligomer is in a food composition.
 73. Themethod of claim 38, wherein the procyanidin oligomer is in a dietarysupplement composition.